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BACKGROUND: Treatment adherence (TA) in narcolepsy is a complex phenomenon influenced by various factors beyond patient-related aspects. The management of narcolepsy involves non-pharmacological and symptomatic pharmacological treatment. Factors such as chronic daytime sleepiness, cognitive deficits, psychiatric comorbidities and adverse effects of pharmacological treatment are aspects of narcolepsy that could undermine TA, impacting patients' ability or willingness to consistently follow treatment plans. The aim of this study was to identify the factors influencing TA in narcolepsy and to determine the most significant barriers to adherence. METHODS: An online survey was conducted during the pandemic, assessing demographic and clinical data, medication usage, and adverse effects of treatment. Various questionnaires, such as the Adherence Barriers Questionnaire (ABQ) and Epworth Sleepiness Scale (ESS), were utilized. The ABQ identified patient-specific barriers to medication adherence, while the Patient Health Questionnaire (PHQ-9) assessed depressive symptoms. RESULTS: We analyzed 243 narcolepsy patients (77 % female, mean age 35.7 ± 12.3 years) with 71 % having narcolepsy type 1 (NT1). The average ESS score was 16.4 (SD ± 3.7). Adherence barriers (AB) were identified in 89 % of patients (216/243) based on ABQ score. The most common barriers reported were "Forgetfulness" (77 %), "Depression" (57 %), and "Side effect-driven medication reduction/stopping behavior" (49 %). Approximately 72 % of patients reported side effects from their narcolepsy medication, leading to discontinuation in 78 % of cases. A moderate correlation was found between the severity of adherence barriers (ABQ score) and levels of depression (PHQ-9 score; rs = 0.412, p = 00.000), as well as ESS score (p = . 048). The results of this study may have been influenced by the pandemic situation. CONCLUSION: Adherence barriers are common (89 %) and diverse among people with narcolepsy. Many barriers are related to excessive daytime sleepiness (EDS), cognitive deficits or depressive symptoms, highlighting the importance of recognizing and addressing them for optimal TA. Medication side effects, especially occurring when polypharmacology is utilized, also significantly contribute to adherence challenges. Effective communication regarding therapy adherence and improved detection and management of EDS and depression are crucial for enhancing TA in narcolepsy patients.
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Cumplimiento de la Medicación , Narcolepsia , Humanos , Narcolepsia/tratamiento farmacológico , Narcolepsia/psicología , Femenino , Masculino , Adulto , Encuestas y Cuestionarios , Cumplimiento de la Medicación/psicología , Cumplimiento de la Medicación/estadística & datos numéricos , Persona de Mediana Edad , Depresión/tratamiento farmacológicoRESUMEN
Despite an unprecedented number of women entering neuroscience, and decades-long recruitment and retention efforts, women continue to be disproportionately underrepresented in European academic tenure-track faculty and leadership positions. This Perspective focuses on two major career points where women exhibit diminished representation: the transition from postdoctoral fellow to junior professor and the promotion to more senior (tenured) faculty positions. We discuss below recently implemented country-specific and Europe-wide initiatives supporting equal career progression and propose further concrete steps to be taken to break down the structural barriers that prevent women's progression up the academic career ladder as European neuroscientists.
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Centros Médicos Académicos , Docentes Médicos , Humanos , Femenino , Movilidad Laboral , LiderazgoRESUMEN
BACKGROUND: Management of narcolepsy includes behavior strategies and symptomatic pharmacological treatment. In the general population, complementary and alternative medicine (CAM) use is common in Europe (30%), also in chronic neurological disorders (10-20%). The aim of our study was to evaluate frequency and characteristics of CAM use in German narcolepsy patients. METHODS: Demographic, disease-related data frequency and impact of CAM use were assessed in an online survey. Commonly used CAM treatments were predetermined in a questionnaire based on the National Center for Complementary and Alternative Medicine and included the domains: (1) alternative medical systems; (2) biologically based therapies; (3) energy therapies; (4) mind-body interventions, and (5) manipulative and body-based therapies. RESULTS: We analyzed data from 254 questionnaires. Fifteen percent of participants were at the time of survey administration using CAM for narcolepsy, and an additional 18% of participants reported past use. Among the 33% of CAM users, vitamins/trace elements (54%), homoeopathy (48%) and meditation (39%) were used most frequently. 54% of the users described CAM as helpful. CAM users more frequently described having side effects from their previous medication (p = 0.001), and stated more frequently not to comply with pharmacological treatment than non-CAM users (21% vs. 8%; p = 0.024). DISCUSSION: The use of CAM in narcolepsy patients is common. Our results indicate that many patients still feel the need to improve their symptoms, sleepiness and psychological well-being in particular. Frequent medication change, the experience of adverse events and low adherence to physician-recommended medication appears more frequent in CAM users. The impact of CAM however seems to be limited.
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Terapias Complementarias , Meditación , Narcolepsia , Humanos , Encuestas y Cuestionarios , Narcolepsia/tratamiento farmacológico , EmocionesRESUMEN
As our understanding of the cell interior has grown, we have come to appreciate that most cellular operations are localized, that is, they occur at discrete and identifiable locations or domains. These cellular domains contain enzymes, machines, and other components necessary to carry out and regulate these localized operations. Here, we review these features of one such operation: the localization and translation of mRNAs within subcellular compartments observed across cell types and organisms. We describe the conceptual advantages and the "ingredients" and mechanisms of local translation. We focus on the nature and features of localized mRNAs, how they travel and get localized, and how this process is regulated. We also evaluate our current understanding of protein synthesis machines (ribosomes) and their cadre of regulatory elements, that is, the translation factors.
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Biosíntesis de Proteínas , Ribosomas , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
How newly synthesized integral membrane proteins and secreted factors are sorted and trafficked to the appropriate location in different cell types remains an important problem in cell biology. One powerful approach for elucidating the trafficking route of a specific protein is to sequester it following synthesis in the endoplasmic reticulum and trigger its release with an externally applied cue. Combined with fluorescent probes, this approach can be used to directly visualize each trafficking step as cargo molecules progress through the different organelles of the secretory network. Here, we discuss design strategies and practical implementation of an inducible protein secretion system we recently developed (zapalog mediated ER trap: zapERtrap) that allows one to use light to initiate secretory trafficking from targeted cells or subcellular domains. We provide detailed protocols for experiments using this approach to visualize protein trafficking from the endoplasmic reticulum to the plasma membrane in fibroblast cell lines and primary cultured neurons.
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Retículo Endoplásmico , Proteínas de la Membrana , Transporte Biológico , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
Binding of two different CaM kinases, CaMKII and DAPK1, to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B near S1303 has been implicated in excitotoxic/ischemic neuronal cell death. The GluN2BΔCaMKII mutation (L1298A, R1300Q) is neuroprotective but abolishes only CaMKII but not DAPK1 binding. However, both kinases can additionally phosphorylate GluN2B S1303. Thus, we here tested S1303 phosphorylation for possible contribution to neuronal cell death. The GluN2BΔCaMKII mutation completely abolished phosphorylation by CaMKII and DAPK1, suggesting that the mutation could mediate neuroprotection by disrupting phosphorylation. However, S1303 phosphorylation was not increased by excitotoxic insults in hippocampal slices or by global cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation in vivo. In hippocampal cultures, S1303 phosphorylation was induced by chemical LTD but not LTP stimuli. These results indicate that the additional effect of the GluN2BΔCaMKII mutation on phosphorylation needs to be considered only in LTD but not in LTP or ischemia/excitotoxicity.
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Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.
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Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Neuronas/metabolismo , Vías Secretoras/genética , Sinapsis/metabolismo , Transmisión Sináptica/genética , Animales , Animales Recién Nacidos , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Membrana Celular/ultraestructura , Retículo Endoplásmico/ultraestructura , Femenino , Colorantes Fluorescentes/química , Expresión Génica , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Hipocampo/citología , Hipocampo/metabolismo , Luz , Masculino , Imagen Molecular/métodos , Neuronas/citología , Cultivo Primario de Células , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Receptores AMPA/genética , Receptores AMPA/metabolismo , Sinapsis/ultraestructura , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision.
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Transporte Axonal , Neuronas/metabolismo , Imagen Óptica/métodos , Animales , Humanos , Neuronas/ultraestructura , Receptores de Neurotransmisores/metabolismoRESUMEN
Four CaMKII isoforms are encoded by distinct genes, and alternative splicing within the variable linker-region generates additional diversity. The α and ß isoforms are largely brain-specific, where they mediate synaptic functions underlying learning, memory and cognition. Here, we determined the α and ß splice-variant distribution among different mouse brain regions. Surprisingly, the nuclear variant αB was detected in all regions, and even dominated in hypothalamus and brain stem. For CaMKIIß, the full-length variant dominated in most regions (with higher amounts of minor variants again seen in hypothalamus and brain stem). The mammalian but not fish CaMKIIß gene lacks exon v3N that encodes the nuclear localization signal in αB, but contains three exons not found in the CaMKIIα gene (exons v1, v4, v5). While skipping of exons v1 and/or v5 generated the minor splice-variants ß', ße and ße', essentially all transcripts contained exon v4. However, we instead detected another minor splice-variant (now termed ßH), which lacks part of the hub domain that mediates formation of CaMKII holoenzymes. Surprisingly, in an optogenetic cellular assay of protein interactions, CaMKIIßH was impaired for binding to the ß hub domain, but still bound CaMKIIα. This provides the first indication for isoform-specific differences in holoenzyme formation.
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Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Holoenzimas/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Exones/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Embarazo , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs) with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca2+ from distinct sources. NMDA receptor Ca2+ initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca2+ channels (L-VGCCs) regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion.
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Dendritas/metabolismo , Potenciación a Largo Plazo/fisiología , Animales , Células Cultivadas , Endosomas/metabolismo , Ratas Sprague-Dawley , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismoRESUMEN
Neurons face the challenge of regulating the abundance, distribution and repertoire of integral membrane proteins within their immense, architecturally complex dendritic arbors. While the endoplasmic reticulum (ER) supports dendritic translation, most dendrites lack the Golgi apparatus (GA), an essential organelle for conventional secretory trafficking. Thus, whether secretory cargo is locally trafficked in dendrites through a non-canonical pathway remains a fundamental question. Here we define the dendritic trafficking itinerary for key synaptic molecules in rat cortical neurons. Following ER exit, the AMPA-type glutamate receptor GluA1 and neuroligin 1 undergo spatially restricted entry into the dendritic secretory pathway and accumulate in recycling endosomes (REs) located in dendrites and spines before reaching the plasma membrane. Surprisingly, GluA1 surface delivery occurred even when GA function was disrupted. Thus, in addition to their canonical role in protein recycling, REs also mediate forward secretory trafficking in neuronal dendrites and spines through a specialized GA-independent trafficking network.
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Moléculas de Adhesión Celular Neuronal/metabolismo , Corteza Cerebral/fisiología , Dendritas/metabolismo , Endosomas/metabolismo , Receptores AMPA/metabolismo , Animales , Transporte de Proteínas , RatasRESUMEN
A DNA vaccine containing the glycoprotein (G) gene of the North American viral hemorrhagic septicemia virus (VHSV) genotype IVb was developed to evaluate the immune response of fish following vaccination and evaluate its efficacy in protecting a susceptible species, the Muskellunge Esox masquinongy, against VHSV-IVb challenge. Seven weeks (539 degree-days) following vaccination with 10 µg of either pVHSivb-G or a control plasmid, Muskellunge were challenged by immersion with 105 plaque-forming units (pfu)/mL of VHSV-IVb. Fish vaccinated with pVHSivb-G had a relative percent survival (RPS) of 45%. Vaccinated fish also had significantly lower mean viral titers in tissues (4.2 × 102 pfu/g) and viral prevalence (4%) than fish receiving the plasmid control vaccine (3.3 × 105 pfu/g; 82%). Neutralizing antibodies were detected 28 d (308 degree-days) postchallenge (11 weeks postvaccination) in 100% of Muskellunge vaccinated with pVHSivb-G compared with only 12% of plasmid-control-vaccinated Muskellunge, suggesting robust induction of a secondary, adaptive immune response. In addition, pVHSivb-G-vaccinated Rainbow Trout Oncorhynchus mykiss challenged 7 d (100 degree-days) postvaccination with the heterologous novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), experienced an RPS of 61%, compared to control fish, suggesting induction of an early and transient nonspecific antiviral immune response. This study provides an important starting point for VHSV-IVb vaccine development and useful information about the antiviral immune response elicited by DNA vaccination in a nondomesticated fish species. Received May 1, 2016; accepted September 1, 2016.
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Esocidae , Enfermedades de los Peces/prevención & control , Septicemia Hemorrágica Viral/inmunología , Vacunación/veterinaria , Animales , ADN , Esocidae/virología , Novirhabdovirus , Oncorhynchus mykissRESUMEN
Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 µM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 µM to 125 ± 40 µM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 µM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.
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Luz , Proteínas de la Membrana/metabolismo , Animales , Células Cultivadas , Dimerización , Cinética , RatonesRESUMEN
Photosynthetic cells of most land plant lineages have numerous small chloroplasts even though most algae, and even the early diverging land plant group the hornworts, tend to have one or a few large chloroplasts. One constraint that small chloroplasts could improve is the resistance to CO2 diffusion from the atmosphere to the chloroplast stroma. We examined the mesophyll conductance (inverse of the diffusion resistance) of mutant Arabidopsis thaliana plants with one or only a few large chloroplasts per cell. The accumulation and replication of chloroplasts (arc) mutants of A. thaliana were studied by model fitting to gas exchange data and (13)CO2 discrimination during carbon fixation. The two methods generally agreed, but the value of the CO2 compensation point of Rubisco (Γ *) used in the model had a large impact on the estimated photosynthetic parameters, including mesophyll conductance. We found that having only a few large chloroplasts per cell resulted in a 25-50 % reduction in the mesophyll conductance at ambient CO2.