Orchestrating a New Path for Multiple Sclerosis: Achieving Physical, Cognitive, and Emotional Rehabilitation Goals Through Physical and Music Therapy.

BACKGROUND: Interdisciplinary therapies for the management of people with multiple sclerosis (MS) are underappreciated. There is an urgent need to introduce music therapy (MT), either alone or in combination with physical therapy (PT), into clinical practice to achieve synergy with disease-modifying therapies. A holistic approach to rehabilitation for people with MS may mitigate symptoms and reduce polypharmacy, potentially lowering health care costs. RESULTS: As MS progresses, patients experience a range of worsening symptoms, and many develop psychosocial comorbidities. As disease-modifying therapies delay disability progression, nonpharmacologic treatments become increasingly important. The main aim of PT is to improve or maintain patients' functional mobility, strength, and flexibility. Because it targets multiple functions, MT can help improve functional and psychosocial domains and may be a valuable intervention to help patients achieve the physical, cognitive, and emotional goals of PT. Exploratory studies showed that MT, alone or in combination with PT, can lead to functional improvements in mobility, balance, gait, and fatigue. Similar to PT, MT also has benefits in improving fine motor skills, cognition, learning, and memory and in providing emotional support. CONCLUSIONS: Both MT and PT have the potential to improve overall well-being and health-related quality of life in physically active patients with MS, and MT can provide added emotional support for those who are less able to engage in physical activity. However, MT is not typically a part of standard of care, and PT visits are limited. Nevertheless, interdisciplinary therapies should be incorporated into clinical practice.

Immune cell subset profiling in multiple sclerosis after fingolimod initiation and continued treatment: The FLUENT study.

Background: Fingolimod is a sphingosine 1-phosphate receptor modulator approved for relapsing MS. Long-term effects on the immunological profile are not fully understood. Objective: Investigate fingolimod's temporal effects on immune cell subsets, and safety outcomes. Methods: In FLUENT, a 12-month, prospective, non-randomized, open-label, phase IV study, adult participants received fingolimod 0.5 mg/day. Changes in immune cell subsets, anti-John Cunningham virus (JCV) antibody index, and serum neurofilament levels were assessed. Results: 165 fingolimod-naive and 217 participants treated for 2-12 years in routine clinical practice were enrolled. Levels of all monitored peripheral lymphocyte subsets were reduced from month 3 in fingolimod-naive participants. Greatest reductions occurred in naive and central memory CD4+ and CD8+ T cells, and in naive and memory B cells. Most lymphocyte subset levels remained stable in the continuous fingolimod group. Components of the innate immune system remained within reference ranges. No increase in JCV seropositivity was observed. No single cellular subset correlated with anti-JCV antibody index at any time point. Neurofilament levels remained within healthy adult reference limits throughout. No opportunistic infections were reported; no new or unexpected safety signals were observed. Conclusion: FLUENT provides insights into the utility of immunological profiling to evaluate therapy response and potential infection risk.

Topical human recombinant nerve growth factor for stage 1 Neurotrophic Keratitis: Retrospective case series of cenegermin treatment.

Purpose: To report on the management and effectiveness of treating patients with stage I Neurotrophic Keratitis using an 8-week course of topical recombinant human nerve growth factor (rhNGF, cenegermin). Observations: In this retrospective case series, punctate epithelial erosions (PEE), best corrected visual acuity (BCVA) and corneal sensation were followed and documented from 2 to 12 months in patients treated as per the standard of care. Clinical outcomes including changes in PEEs, corneal sensation and BCVA are reported. Most patients also had preexisting thyroid disease. Conclusions: All patients had clinically significant improvements in PEE, and corneal sensation. Three of the four patients had a significant improvement in BCVA, one patient had no change in their pre-treatment visual acuity (BCVA 20/20) The four patients studied also reported decreased photophobia and improvements in their quality of life. This case series provides real-world evidence of the safety and efficacy of cenegermin treatment of stage I NK for all four patients.

A Targeted Literature Search and Phenomenological Review of Perspectives of People with Multiple Sclerosis and Healthcare Professionals of the Immunology of Disease-Modifying Therapies.

INTRODUCTION: The mechanisms of action of disease-modifying therapies (DMTs) for multiple sclerosis (MS) are complex and involve an interplay of immune system components. People with MS (PwMS) may lack a clear understanding of the immunological pathways involved in MS and its treatment; effective communication between healthcare professionals (HCPs) and PwMS is needed to facilitate shared decision-making when discussing the disease and selecting DMTs and is particularly important in the coronavirus disease 2019 (COVID-19) era. METHODS: In this patient-authored two-part review, we performed a targeted literature search to assess the need for better communication between HCPs and PwMS regarding treatment selection, and also conducted a qualitative survey of four patient and care-partner authors to obtain insights regarding their understanding of and preferences for the treatment and management of MS. RESULTS: Following a search of the Embase and MEDLINE databases using Ovid in June 2020, an analysis of 40 journal articles and conference abstracts relating to patient empowerment and decision-making in DMT selection for MS showed a preference for safety and efficacy of treatments, followed by autonomy and convenience of administration. A need for better communication between HCPs and PwMS during treatment selection to improve patient satisfaction was also identified. The open survey responses from the patient authors revealed a need for greater involvement in decision-making processes and desire for improved communication and information tools. CONCLUSIONS: This targeted literature search and phenomenological review confirms PwMS preferences for empowered decision-making in disease management and treatment selection, to optimize independence, safety, and efficacy. It also identifies an unmet need for improved communication and information tools that convey MS information in a relatable manner. Furthermore, this review seeks to address this unmet need by providing plain language figures and descriptions of MS immune mechanisms that can be used to facilitate discussions between HCPs and PwMS.

In multiple sclerosis (MS), there are different cells in the immune system that contribute to the disease. The main cells in the immune system are T and B cells. People with MS (PwMS) might not be familiar with details about the immune system, and healthcare professionals might not always communicate details about how treatments work clearly to PwMS when choosing treatments with them. It is important for PwMS to have all the information they need to help make decisions about treatments. This information needs to be given in a way they can understand. This is especially important during the coronavirus disease 2019 (COVID-19) pandemic. In this paper, we first looked at what research has already been published about what is most important to PwMS when making treatment decisions. The existing research says that safety and effectiveness are the most important things and that PwMS prefer treatments that they can take themselves. PwMS also need better communication and information from doctors to make decisions and to help explain how MS treatments work in the body. Next, we gave a survey to the patients who are authors of this paper to ask about what is important to them when making treatment decisions. Their answers were very similar to the existing research. Overall, PwMS need better communication from healthcare professionals about the immune system. This paper also includes plain language descriptions and figures to help healthcare professionals explain and discuss the importance of the immune system in MS with PwMS.

Erenumab dosage for migraine prevention: An evidence-based narrative review with recommendations.

BACKGROUND: Therapeutic monoclonal antibodies against the calcitonin gene-related peptide (CGRP) receptor or its ligand have changed the landscape of treatment options for migraine. Erenumab is the first and only fully human monoclonal antibody designed to target and block the CGRP receptor. It is approved by the Food and Drug Administration for preventive treatment of migraine in adults. The recommended dose of erenumab is 70 mg monthly, with guidance that some patients may benefit from the 140 mg monthly dose. There is a need for information to guide clinical practice on the comparative efficacy and safety of these two dosing options. OBJECTIVE: To evaluate therapeutic and tolerability differences between erenumab 70 and 140 mg based on evidence from published literature. METHODS: This narrative review evaluates therapeutic and tolerability differences between erenumab 70 and 140 mg based on a literature search using PubMed interface, Embase and Ovid MEDLINE(R) databases. The key search terms included migraine, AMG 334, AMG334, erenumab, erenumab-aooe, and Aimovig. The search was limited to English language articles or conference abstracts published up to May 2021. RESULTS: From the literature search, we retrieved 23 relevant articles/conference abstracts (19 articles [5 randomized, double-blind studies] and 4 conference abstracts) for inclusion in this narrative review. Although the recommended starting dosage of erenumab is 70 mg, this narrative review of the literature indicates that some patients may benefit from a dosage of 140 mg erenumab once monthly-especially those with difficult-to-treat disease and prior treatment failures. The evidence indicates that erenumab at 140 mg has a numerically better efficacy than 70 mg across a broad spectrum of migraine outcomes, including preventing progression to chronic migraine. CONCLUSION: Cumulative data from the literature support a therapeutic gain with an increase from erenumab 70 to 140 mg and a rationale for initiating 140 mg in selected patients.

Switching to fingolimod in PREFERMS: Effect of treatment history and naïvety on clinical, MRI and treatment satisfaction outcomes✰.

BACKGROUND: Injectable disease-modifying therapies (iDMTs) are often used as first-line treatments for relapsing multiple sclerosis. Fingolimod is frequently used following treatment with iDMTs. Whether prior iDMT treatment impacts the effectiveness of subsequent fingolimod therapy is unclear. Here, we assessed switching from iDMTs to fingolimod, and the impact of treatment history on fingolimod escalation using data from the 12-month 'Prospective, Randomized, active-controlled, open-label study to Evaluate patient retention on Fingolimod versus approved first-line disease-modifying thErapies in adults with Relapsing-remitting Multiple Sclerosis' (PREFERMS). The study design and results at the end of randomized treatment (EoRT) in PREFERMS have been published. METHODS: Both treatment-naïve patients and those who had previously received an iDMT were eligible for enrolment in PREFERMS, and one treatment switch was permitted on study. Pre-specified exploratory analyses compared outcomes in those randomized to fingolimod or to an iDMT at end of study (EoS), which included time spent on randomized and on switch treatment. Post hoc exploratory analyses (unadjusted for multiplicity owing to the large number of comparisons) among patients randomized to an iDMT who switched to fingolimod, compared outcomes longitudinally before (EoRT) and after (EoS) switching, and compared outcomes at EoRT and EoS among subgroups stratified by iDMT-treatment history. Outcomes included brain volume, various measures of gadolinium-enhancing [Gd+] lesion counts, annualized relapse rate (ARR), Symbol Digit Modalities Test (SDMT) score, patient-reported treatment satisfaction using the Medication Satisfaction Questionnaire (MSQ) and adverse event (AE) rates. RESULTS: At EoS, 255 of 439 patients randomized to an iDMT had switched to fingolimod and 27 of 436 patients randomized to fingolimod had switched to an iDMT. By EoS, 44.2% of total treatment exposure in the iDMT group was to fingolimod and the mean time spent on fingolimod in this group was 220 days (approximately 7 months). Outcomes in the fingolimod group at EoS (brain volume, changes in Gd+ lesion counts, ARR, oral SDMT score and MSQ score) were similar to those seen at EoRT, but in the iDMT group these outcomes were more favorable at EoS than at EoRT and were similar to rates seen in the fingolimod group. Among patients who switched from iDMT to fingolimod, there were longitudinal improvements in ARR (EoRT, 0.3 [95% confidence interval (CI), 0.2-0.4]; EoS, 0.2 [0.1-0.3]; odds ratio, 0.5 [0.3-0.9]) and in treatment satisfaction (proportion of patients with MSQ > 5; EoRT, 67.4%; EoS, 90.4%; odds ratio, 5.7 [95% CI, 3.4-9.4]) after fingolimod treatment, and changes in brain volume, Gd+ lesion count, and AEs or AEs causing discontinuation were also more favorable at EoS than at EoRT. In all patient groups stratified by iDMT-treatment history, differences in outcomes narrowed or disappeared after fingolimod treatment. CONCLUSION: These analyses indicate that patients in PREFERMS had improved outcomes within months of switching to fingolimod from an iDMT and that improvements occurred irrespective of the number of iDMTs previously administered. These data provide a unique opportunity to explore clinical, radiological and safety outcomes associated with a range of clinically relevant treatment pathways.

Correction to: Analysis of cardiac monitoring and safety data in patients initiating fingolimod treatment in the home or in clinic.

Following publication of the original article [1], the authors reported a mistake regarding the year found in the paragraph of the Background section.

Analysis of cardiac monitoring and safety data in patients initiating fingolimod treatment in the home or in clinic.

BACKGROUND: Fingolimod (Gilenya®) is approved for relapsing forms of multiple sclerosis in the USA. Owing to transient heart-rate effects when initiating fingolimod, eligible patients undergo precautionary baseline assessment and first-dose observation (FDO) for ≥6 h. Prior to 2014, FDO was undertaken only in clinics. As the FDO period is short, and fingolimod has accumulated evidence of a positive benefit:risk ratio, an in-home treatment-initiation program, Gilenya@Home, was developed to offer a convenient alternative. METHODS: Cardiac parameters and adverse events (AEs) were recorded by healthcare professionals performing fingolimod FDOs in the US Gilenya@Home program or in US Gilenya Assessment Network clinics. Anonymized data were collated retrospectively from the first 34 months in the home setting and from 78 months in clinics; data are reported descriptively. Satisfaction with Gilenya@Home was rated by patients using a 7-item questionnaire that considered aspects such as ease of scheduling, courtesy, and competency. RESULTS: Data were captured as part of standard care from 5573 patients initiating fingolimod in-home (October 2014 to July 2017) and from 15,025 patients initiating in-clinic (July 2010 to December 2016). In the Gilenya@Home questionnaire, 91.7% of 1848 respondents rated their overall satisfaction as "very good," and 7.6% rated their satisfaction as "good." AEs were reported for 30.7 and 32.6% of in-home and in-clinic patients, respectively. In total, 557 in-home (10.0%) and 398 in-clinic (2.6%) patients were monitored for > 6 h; 15 (0.3%) in-home and 129 (0.9%) in-clinic patients were transferred to an emergency room for overnight monitoring. The mean (standard deviation) heart rate (HR; bpm) pre-FDO was 74.8 (12.2) in-home and 74.2 (11.3) in-clinic; reduction in HR at 6 h postdose was 10.6 (12.0) and 6.3 (9.6), respectively. New-onset first-degree atrioventricular block was experienced by 132 (2.4%) in-home and 74 (0.5%) in-clinic patients, and Wenckebach (Mobitz type I) second-degree atrioventricular block by four (0.07%) and nine (0.1%) patients, with no cases of third-degree atrioventricular block. CONCLUSIONS: A substantial number of patients have initiated fingolimod at home, reporting very high levels of satisfaction. Gilenya@Home was as rigorous as the clinic setting in detecting cardiovascular events. Overall, FDO safety outcomes were similar with Gilenya@Home and in-clinic.

Ca(2+) signaling mechanisms in bovine adrenal chromaffin cells.

Calcium (Ca(2+)) is a crucial intracellular messenger in physiological aspects of cell signaling. Adrenal chromaffin cells are the secretory cells from the adrenal gland medulla that secrete catecholamines, which include epinephrine and norepinephrine important in the 'fight or flight' response. Bovine adrenal chromaffin cells have long been used as an important model for secretion -(exocytosis) not only due to their importance in the short-term stress response, but also as a neuroendocrine model of neurotransmtter release, as they have all the same exocytotic proteins as neurons but are easier to prepare, culture and use in functional assays. The components of the Ca(2+) signal transduction cascade and it role in secretion has been extensively characterized in bovine adrenal chromaffin cells. The Ca(2+) sources, signaling molecules and how this relates to the short-term stress response are reviewed in this book chapter in an endeavor to generally -overview these mechanisms in a concise and uncomplicated manner.

Neuronal calcium sensor-1 regulation of calcium channels, secretion, and neuronal outgrowth.

Calcium (Ca(2+)) is an important intracellular messenger underlying cell physiology. Ca(2+) channels are the main entry route for Ca(2+) into excitable cells, and regulate processes such as neurotransmitter release and neuronal outgrowth. Neuronal Calcium Sensor-1 (NCS-1) is a member of the Calmodulin superfamily of EF-hand Ca(2+) sensing proteins residing in the subfamily of NCS proteins. NCS-1 was originally discovered in Drosophila as an overexpression mutant (Frequenin), having an increased frequency of Ca(2+)-evoked neurotransmission. NCS-1 is N-terminally myristoylated, can bind intracellular membranes, and has a Ca(2+) affinity of 0.3 µM. Over 10 years ago it was discovered that NCS-1 overexpression enhances Ca(2+)-evoked secretion in bovine adrenal chromaffin cells. The mechanism was unclear, but there was no apparent direct effect on the exocytotic machinery. It was revealed, again in chromaffin cells, that NCS-1 regulates voltage-gated Ca(2+) channels (Cavs) in G-Protein Coupled Receptor (GPCR) signaling pathways. This work in chromaffin cells highlighted NCS-1 as an important modulator of neurotransmission. NCS-1 has since been shown to regulate and/or directly interact with many proteins including Cavs (P/Q, N, and L), TRPC1/5 channels, GPCRs, IP3R, and PI4 kinase type IIIß. NCS-1 also affects neuronal outgrowth having roles in learning and memory affecting both short- and long-term synaptic plasticity. It is not known if NCS-1 affects neurotransmission and synaptic plasticity via its effect on PIP2 levels, and/or via a direct interaction with Ca(2+) channels or their signaling complexes. This review gives a historical account of NCS-1 function, examining contributions from chromaffin cells, PC12 cells and other models, to describe how NCS-1's regulation of Ca(2+) channels allows it to exert its physiological effects.

Metabotropic glutamate receptor-mediated LTD involves two interacting Ca(2+) sensors, NCS-1 and PICK1.

There are two major forms of long-term depression (LTD) of synaptic transmission in the central nervous system that require activation of either N-methyl-D-aspartate receptors (NMDARs) or metabotropic glutamate receptors (mGluRs). In synapses in the perirhinal cortex, we have directly compared the Ca(2+) signaling mechanisms involved in NMDAR-LTD and mGluR-LTD. While both forms of LTD involve Ca(2+) release from intracellular stores, the Ca(2+) sensors involved are different; NMDAR-LTD involves calmodulin, while mGluR-LTD involves the neuronal Ca(2+) sensor (NCS) protein NCS-1. In addition, there is a specific requirement for IP3 and PKC, as well as protein interacting with C kinase (PICK-1) in mGluR-LTD. NCS-1 binds directly to PICK1 via its BAR domain in a Ca(2+)-dependent manner. Furthermore, the NCS-1-PICK1 association is stimulated by activation of mGluRs, but not NMDARs, and introduction of a PICK1 BAR domain fusion protein specifically blocks mGluR-LTD. Thus, NCS-1 plays a distinct role in mGluR-LTD.

Differential regulation of endogenous N- and P/Q-type Ca2+ channel inactivation by Ca2+/calmodulin impacts on their ability to support exocytosis in chromaffin cells.

P/Q-type (Ca(V)2.1) and N-type (Ca(V)2.2) Ca2+ channels are critical to stimulus-secretion coupling in the nervous system; feedback regulation of these channels by Ca2+ is therefore predicted to profoundly influence neurotransmission. Here we report divergent regulation of Ca2+-dependent inactivation (CDI) of native N- and P/Q-type Ca2+ channels by calmodulin (CaM) in adult chromaffin cells. Robust CDI of N-type channels was observed in response to prolonged step depolarizations, as well as repetitive stimulation with either brief step depolarizations or action potential-like voltage stimuli. Adenoviral expression of Ca2+-insensitive calmodulin mutants eliminated CDI of N-type channels. This is the first demonstration of CaM-dependent CDI of a native N-type channel. CDI of P/Q-type channels was by comparison modest and insensitive to expression of CaM mutants. Cloning of the C terminus of the Ca(V)2.1 alpha1 subunit from chromaffin cells revealed multiple splice variants lacking structural motifs required for CaM-dependent CDI. The physiological relevance of CDI on stimulus-coupled exocytosis was revealed by combining perforated-patch voltage-clamp recordings of pharmacologically isolated Ca2+ currents with membrane capacitance measurements of exocytosis. Increasing stimulus intensity to invoke CDI resulted in a significant decrease in the exocytotic efficiency of N-type channels compared with P/Q-type channels. Our results reveal unexpected diversity in CaM regulation of native Ca(V)2 channels and suggest that the ability of individual Ca2+ channel subtypes to undergo CDI may be tailored by alternative splicing to meet the specific requirements of a particular cellular function.

Calcium-sensing mechanism in TRPC5 channels contributing to retardation of neurite outgrowth.

The calcium- and sodium-permeable transient receptor potential channel TRPC5 has an inhibitory role in neuronal outgrowth but the mechanisms governing its activity are poorly understood. Here we propose a mechanism involving the neuronal calcium sensor-1 (NCS-1) protein. Inhibitory mutants of TRPC5 and NCS-1 enhance neurite outgrowth similarly. Mutant NCS-1 does not inhibit surface-expression of TRPC5 but generally suppresses channel activity, irrespective of whether it is evoked by carbachol, store depletion, lanthanides or elevated intracellular calcium. NCS-1 and TRPC5 are in the same protein complex in rat brain and NCS-1 directly binds to the TRPC5 C-terminus. The data suggest protein-protein interaction between NCS-1 and TRPC5, and involvement of this protein complex in retardation of neurite outgrowth.

Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions.

We report the protein isolation, cloning and characterization of members of an unusual protein family, which comprise the most abundant proteins present in the squid eye. The proteins in this family have a range of molecular weights from 32 to 36 kDa. Electron microscopy and detergent solubilization demonstrate that these proteins are tightly associated with membrane structures where they may form tetramers. Despite this, these proteins have no stretches of hydrophobic residues that could form typical transmembrane domains. They share an unusual protein sequence rich in methionine, and contain multiple repeating motifs. We have therefore named these proteins Methionine-Rich Repeat Proteins (MRRPs). The use of structure prediction algorithms suggest very little recognized secondary structure elements. At the time of cloning no sequence or structural homologues have been found in any database. We have isolated three closely related cDNA clones from the MRRP family. Coupled in vitro transcription/translation of the MRRP clones shows that they encode proteins with molecular masses similar to components of native MRRPs. Immunoblot analysis of these proteins reveals that they are also present in squid brain, optic lobe, and heart, and also indicate that MRRP-like protein motifs may also exist in mammalian tissues. We propose that MRRPs define a family of important proteins that have an unusual mode of attachment or insertion into cell membranes and are found in evolutionarily diverse organisms.

IL1 receptor accessory protein like, a protein involved in X-linked mental retardation, interacts with Neuronal Calcium Sensor-1 and regulates exocytosis.

Previously, human genetics-based approaches allowed us to show that mutations in the IL-1 receptor accessory protein-like gene (IL1RAPL) are responsible for a non-specific form of X-linked mental retardation. This gene encodes a predicted protein of 696 amino acids that belongs to a novel class of the IL-1/Toll receptor family. In addition to the extracellular portion consisting of three Ig-like domains and the intracellular TIR domain characteristic of the IL-1/Toll receptor family, IL1RAPL contains a specific 150 amino acid carboxy terminus that has no significant homology with any protein of known function. In order to begin to elucidate the function of this IL-1/Toll receptor-like protein, we have assessed the effect of recombinant IL1RAPL on the binding affinity of type I IL-1R for its ligands IL-1alpha and beta and searched for proteins interacting with the specific carboxy terminus domain of IL1RAPL. Our results show that IL1RAPL is not a protein receptor for IL-1. In addition we present here the identification of Neuronal Calcium Sensor-1 (NCS-1) as an IL1RAPL interactor. Remarkably, although NCS-1 and its non-mammalian homologue, frequenin, are members of a highly conserved EF-hand Ca(2+) binding protein family, our data show that IL1RAPL interacts only with NCS-1 through its specific C-terminal domain. The functional relevance of IL1RAPL activity was further supported by the inhibitory effect on exocytosis in PC12 cells overexpressing IL1RAPL. Taken together, our data suggest that IL1RAPL may regulate calcium-dependent exocytosis and provide insight into the understanding of physiopathological mechanisms underlying cognitive impairment resulting from IL1RAPL dysfunction.

Sense and sensibility in the regulation of voltage-gated Ca(2+) channels.

Voltage-gated Ca(2+) channels are crucial for neurotransmitter release and other neuronal functions, and their activity-dependent regulation could underlie various aspects of synaptic plasticity. Recent studies have identified Ca(2+)-sensing proteins involved in Ca(2+)-channel modulation. These have complex effects on channel gating, and data suggest that the actions of multiple Ca(2+) sensors are important for the fine-tuning of Ca(2+) channel activity.

Identification of Ca2+-dependent binding partners for the neuronal calcium sensor protein neurocalcin delta: interaction with actin, clathrin and tubulin.

The neuronal calcium sensors are a family of EF-hand-containing Ca(2+)-binding proteins expressed predominantly in retinal photoreceptors and neurons. One of the family members is neurocalcin delta, the function of which is unknown. As an approach to elucidating the protein interactions made by neurocalcin delta, we have identified brain cytosolic proteins that bind to neurocalcin delta in a Ca(2+)-dependent manner. We used immobilized recombinant myristoylated neurocalcin delta combined with protein identification using MS. We demonstrate a specific interaction with clathrin heavy chain, alpha- and beta-tubulin, and actin. These interactions were dependent upon myristoylation of neurocalcin delta indicating that the N-terminal myristoyl group may be important for protein-protein interactions in addition to membrane association. Direct binding of neurocalcin delta to clathrin, tubulin and actin was confirmed using an overlay assay. These interactions were also demonstrated for endogenous neurocalcin delta by co-immunoprecipitation from rat brain cytosol. When expressed in HeLa cells, neurocalcin delta was cytosolic at resting Ca(2+) levels but translocated to membranes, including a perinuclear compartment (trans-Golgi network) where it co-localized with clathrin, following Ca(2+) elevation. These data suggest the possibility that neurocalcin delta functions in the control of clathrin-coated vesicle traffic.

Differential use of myristoyl groups on neuronal calcium sensor proteins as a determinant of spatio-temporal aspects of Ca2+ signal transduction.

The localizations of three members of the neuronal calcium sensor (NCS) family were studied in HeLa cells. Using hippocalcin-EYFP and NCS-1-ECFP, it was found that their localization differed dramatically in resting cells. NCS-1 had a distinct predominantly perinuclear localization (similar to trans-Golgi markers), whereas hippocalcin was present diffusely throughout the cell. Upon the elevation of intracellular Ca(2+), hippocalcin rapidly translocated to the same perinuclear compartment as NCS-1. Another member of the family, neurocalcin delta, also translocated to this region after a rise in Ca(2+) concentration. Permeabilization of transfected cells using digitonin caused loss of hippocalcin and neurocalcin delta in the absence of calcium, but in the presence of 10 microm Ca(2+), both proteins translocated to and were retained in the perinuclear region. NCS-1 localization was unchanged in permeabilized cells regardless of calcium concentration. The localization of NCS-1 was unaffected by mutations in all functional EF hands, indicating that its localization was independent of Ca(2+). A minimal myristoylation motif (hippocalcin-(1-14)) fused to EGFP resulted in similar perinuclear targeting, showing that localization of these proteins is because of the exposure of the myristoyl group. This was confirmed by mutation of the myristoyl motif of NCS-1 and hippocalcin that resulted in both proteins remaining cytosolic, even at elevated Ca(2+) concentration. Dual imaging of hippocalcin-EYFP and cytosolic Ca(2+) concentration in Fura Red-loaded cells demonstrated the kinetics of the Ca(2+)/myristoyl switch in living cells and showed that hippocalcin rapidly translocated with a half-time of approximately 12 s after a short lag period when Ca(2+) was elevated. These results demonstrate that closely related Ca(2+) sensor proteins use their myristoyl groups in distinct ways in vivo in a manner that will determine the time course of Ca(2+) signal transduction.