Real-World Adherence to OnabotulinumtoxinA Treatment for Spasticity: Insights From the ASPIRE Study.

Abstract Objective To identify baseline characteristics and treatment-related variables that affect adherence to onabotulinumtoxinA treatment from the Adult Spasticity International Registry (ASPIRE) study. Design Prospective, observational registry (NCT01930786). Setting International clinical sites. Participants Adults with spasticity (N=730). Interventions OnabotulinumtoxinA at clinician's discretion. Main Outcome Measures Clinically meaningful thresholds used for treatment adherent (≥3 treatment sessions during 2-year study) and nonadherent (≤2 sessions). Data analyzed using logistic regression and presented as odds ratios (ORs) with 95% confidence intervals (CIs). Treatment-related variables assessed at sessions 1 and 2 only. Results Of the total population, 523 patients (71.6%) were treatment adherent with 5.3±1.6 sessions and 207 (28.4%) were nonadherent with 1.5±0.5 sessions. In the final model (n=626/730), 522 patients (83.4%) were treatment adherent and 104 (16.6%) were nonadherent. Baseline characteristics associated with adherence: treated in Europe (OR=1.84; CI, 1.06-3.21; P=.030) and use of orthotics (OR=1.88; CI, 1.15-3.08; P=.012). Baseline characteristics associated with nonadherence: history of diplopia (OR=0.28; CI, 0.09-0.89; P=.031) and use of assistive devices (OR=0.51; CI, 0.29-0.90; P=.021). Treatment-related variables associated with nonadherence: treatment interval ≥15 weeks (OR=0.43; CI, 0.26-0.72; P=.001) and clinician dissatisfaction with onabotulinumtoxinA to manage pain (OR=0.18; CI, 0.05-0.69; P=.012). Of the population with stroke (n=411), 288 patients (70.1%) were treatment adherent with 5.3±1.6 sessions and 123 (29.9%) were nonadherent with 1.5±0.5 session. In the final stroke model (n=346/411), 288 patients (83.2%) were treatment adherent and 58 (16.8%) were nonadherent. Baseline characteristics associated with adherence: treated in Europe (OR=2.99; CI, 1.39-6.44; P=.005) and use of orthotics (OR=3.18; CI, 1.57-6.45; P=.001). Treatment-related variables associated with nonadherence: treatment interval ≥15 weeks (OR=0.42; CI, 0.21-0.83; P=.013) and moderate/severe disability on upper limb Disability Assessment Scale pain subscale (OR=0.40; CI, 0.19-0.83; P=.015). Conclusions These ASPIRE analyses demonstrate real-world patient and clinical variables that affect adherence to onabotulinumtoxinA and provide insights to help optimize management strategies to improve patient care

Modification of a Hydrophobic Layer by a Point Mutation in Syntaxin 1A Regulates the Rate of Synaptic Vesicle Fusion

Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex

Diacylglycerol, novel protein kinase C isozymes [eta] and [theta], and other diacylglycerol activated proteins promote neuroprotective plasmalemmal sealing in B104 neurons in vitro and rat sciatic nerve axons in vivo

textTo survive, neurons and other eukaryotic cells must rapidly repair (seal) plasmalemmal damage. Such repair occurs by an accumulation of intracellular vesicles at or near the plasmalemmal disruption. Diacylglycerol (DAG)-dependent and cAMP-dependent proteins are involved in many vesicle trafficking pathways. Although recent studies have implicated the signaling molecule cAMP in sealing, no study has investigated how DAG and DAG-dependent proteins affect sealing and, whether pharmacological inhibition of such proteins could promote immediate repair of damaged mammalian axons. To this end, I investigated the role of DAG, protein kinase C (PKC) and other DAG-activated proteins in plasmalemmal sealing in B104 neurons in vitro and rat sciatic nerves in vivo. Using dye exclusion to assess Ca2+-dependent vesicle-mediated sealing of transected neurites of individually identifiable rat hippocampal B104 cells, I now report that, compared to non-treated controls, sealing probabilities and rates are increased by DAG and cAMP analogs that activate PKC and Munc13-1, and protein kinase A (PKA). Sealing is decreased by inhibiting DAG-activated novel protein kinase C isozymes η (nPKCη) and θ (nPKCθ) and, Munc13-1, the PKC effector myristoylated alanine rich PKC substrate (MARCKS) or phospholipase C (PLC). DAG-increased sealing is prevented by inhibiting MARCKS or PKA. Sealing probability is further decreased by simultaneously inhibiting nPKCη, nPKCθ and PKA. Extracellular Ca2+, DAG or cAMP analogs do not affect this decrease in sealing. I also report that applying inhibitors of nPKC and PKA to rat sciatic axons crush-severed in vivo under physiological calcium, do not promote immediate repair by polyethylene glycol (PEG), as assessed by compound action potential conduction and dye diffusion through crush sites. These and other data suggest that DAG increases sealing through MARCKS and that nPKCη, nPKCθ and PKA are all required to seal plasmalemmal damage in B104 neurons, and likely all eukaryotic cells.Cellular and Molecular Biolog

OnabotulinumtoxinA Dosing, Disease Severity, and Treatment Benefit in Patients With Cervical Dystonia: A Cohort Analysis From CD PROBE

Introduction: The Cervical Dystonia Patient Registry for Observation of OnabotulinumtoxinA Efficacy (CD PROBE) study ( identifier: NCT00836017), a multicenter, prospective, observational registry, was designed to identify real-world practices and outcomes for patients with cervical dystonia (CD) treated with onabotulinumtoxinA (onabotA). This secondary analysis from CD PROBE aims to determine the impact of presentation subtype on onabotA utilization and CD severity. Materials and Methods: The study cohort includes those who completed all 3 treatments, 4 office visits, and had data recorded for all assessments. Patient outcomes were assessed with the Cervical Dystonia Impact Profile (CDIP-58), Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS), and determination of CD severity. Treatment interval, dose, and adverse events (AEs) were also recorded. Data were stratified according to prior exposure to botulinum toxins (BoNTs) and analyzed with descriptive statistics. Results: Torticollis was the most common presentation subtype in the study cohort (N = 350); the proportion of patients with torticollis was highest in those with severe disease. At each treatment, between 40.7 and 65.2% of those categorized as severe shifted to moderate or mild severity after treatment. Sustained improvements in CDIP-58 and TWSTRS were observed regardless of prior exposure to BoNTs. Dosing of onabotA generally increased from injection 1 to injection 3 and tended to be lower for patients naive to BoNT. Median time interval between injections for the study cohort was 94.0 to 97.5 days. The most common AEs (dysphagia, muscular weakness) and injection intervals were similar between naive vs. non-naive patients; there were no serious treatment-related AEs. Conclusions: This secondary cohort analysis from CD PROBE demonstrates that three repeat treatments with onabotA at intervals consistent with labeling attenuated disease severity and neck pain, resulting in sustained improvements in physician- and patient-reported outcomes. No new safety signals were identified

A model for sealing plasmalemmal damage in neurons and other eukaryotic cells

Plasmalemmal repair is necessary for survival of damaged eukaryotic cells. Ca2 influx through plasmalemmal disruptions activates calpain, vesicle accumulation at lesion sites, and membrane fusion proteins; Ca2 influx also initiates competing apoptotic pathways. Using the formation of a dye barrier (seal) to assess plasmalemmal repair, we now report that B104 hippocampal cells with neurites transected nearer ( 50 m)to the soma seal at a lower frequency and slower rate compared to cells with neurites transected farther ( 50 m) from the soma. Analogs of cAMP, including protein kinase A (PKA)-specific and Epac-specific cAMP, each increase the frequency and rate of sealing and can even initiate sealing in the absence of Ca2 influx at both transection distances. Furthermore, Epac activates a cAMP-dependent, PKA-independent, pathway involved in plasmalemmal sealing. The frequency and rate of plasmalemmal sealing are decreased by a small molecule inhibitor of PKA targeted to its catalytic subunit (KT5720), a peptide inhibitor targeted to its regulatory subunits (PKI), an inhibitor of a novel PKC (an nPKC pseudosubstrate fragment), and an antioxidant (melatonin). Given these and other data, we propose a model for redundant parallel pathways of Ca2 -dependent plasmalemmal sealing of injured neurons mediated in part by nPKCs, cytosolic oxidation, and cAMP activation of PKA and Epac. We also propose that the evolutionary origin of these pathways and substances was to repair plasmalemmal damage in eukaryotic cells. Greater understanding of vesicle interactions, proteins, and pathways involved in plasmalemmal sealing should suggest novel neuroprotective treatments for traumatic nerve injuries and neurodegenerative disorders.This work was funded by grants from the Lone Star Paralysis Foundation.Neuroscienc

Levetiracetam Differentially Alters CD95 Expression of Neuronal Cells and the Mitochondrial Membrane Potential of Immune and Neuronal Cells in vitro

Epilepsy is a neurological seizure disorder that affects over 100 million people worldwide. Levetiracetam, either alone, as monotherapy, or as adjunctive treatment, is widely used to control certain types of seizures. Despite its increasing popularity as a relatively safe and effective anti-convulsive treatment option, its mechanism(s) of action are poorly understood. Studies have suggested neuronal, glial, and immune mechanisms of action. Understanding the precise mechanisms of action of levetiracetam would be extremely beneficial in helping to understand the processes involved in seizure generation and epilepsy. Moreover, a full understanding of these mechanisms would help to create more efficacious treatments while minimizing side-effects. The current study examined the effects of levetiracetam on the mitochondrial membrane potential of neuronal and non-neuronal cells, in vitro, in order to determine if levetiracetam influences metabolic processes in these cell types. In addition, this study sought to address possible immune-mediated mechanisms by determining if levetiracetam alters the expression of immune receptor–ligand pairs. The results show that levetiracetam induces expression of CD95 and CD178 on NGF-treated C17.2 neuronal cells. The results also show that levetiracetam increases mitochondrial membrane potential on C17.2 neuronal cells in the presence of nerve growth factor. In contrast, levetiracetam decreases the mitochondrial membrane potential of splenocytes and this effect was dependent on intact invariant chain, thus implicating immune cell interactions. These results suggest that both neuronal and non-neuronal anti-epileptic activities of levetiracetam involve control over energy metabolism, more specifically, mΔΨ. Future studies are needed to further investigate this potential mechanism of action

Conservation and Divergence of Threonine 254 among Different Syntaxin Orthologs

<div><p>(A) Proposed model of SNARE complex assembly and disassembly in a synaptic vesicle cycle (adapted from [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b005" target="_blank">5</a>]). (1) Synaptobrevin forms a partial <i>trans</i> SNARE complex with syntaxin 1A and SNAP-25. (2) By zippering in an N- to C-termini direction, the SNARE proteins form a <i>trans</i> complex and bring the synaptic vesicle close to the plasma membrane. SNARE-mediated synaptic vesicle exocytosis occurs either spontaneously (3) or evoked by Ca²⁺ (4). (5) <i>cis</i> SNARE complexes are thought to be disassembled by NSF ATPase prior to vesicle recycling. ER, endoplasmic reticulum; PM, plasma membrane; SV, synaptic vesicle.</p> <p>(B) Alignment of amino acids (aa) around position T254 in the <i>Drosophila</i> syntaxin 1A or equivalent residues in syntaxin orthologs from a variety of animals, yeast, and the plant <i>Arabidopsis</i>. The top panel shows a cartoon of syntaxin 1A and the region of the alignment. Syntaxins are organized as “plasma membrane” or “intracellular compartments” according to their cellular distributions. With the exception of syntaxin 4, most plasma membrane syntaxins are known to function in presynaptic terminals or neurosecretory cells for Ca²⁺-regulated exocytosis. Note that T254 is highly conserved among “presynaptic” syntaxin 1A, 2, and 3A molecules. We call all other syntaxin orthologs shown here “constitutive” syntaxins because they are used for constitutive secretion on the plasma membrane (PM) and intracellular compartments, such as the endosome and the lysosome, the <i>cis</i> and <i>trans</i> Golgi network (Golgi network), and endoplasmic reticulum (ER) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-b001" target="_blank">1</a>]. The yeast plasma membrane syntaxin orthologs SSO1 and SSO2, and syntaxins 4 and 131 from <i>Arabidopsis</i> are also shown here. (A more complete alignment can be see in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050072#pbio-0050072-sg001" target="_blank">Figure S1</a>.) Unlike the synaptic syntaxins, syntaxin 4 and most syntaxin 11s have a valine (V) at the 254 equivalent position, syntaxins 6, 7, 12, 16, and 17 a leucine (L), and syntaxin 5 an isoleucine (I). The isoleucine found in the <i>syx^3–69</i> mutant resembles some of the wild-type syntaxin orthologs used for constitutive secretion. The core complex layers from 0 to +8 are identified at the bottom. The aa sequence was obtained from the NIH's National Center for Biotechnology Information (NCBI; <a href="http://www.ncbi.nlm.nih.gov" target="_blank">http://www.ncbi.nlm.nih.gov</a>) and aligned using the software DNAStar.</p></div

The T254I Mutation Exerts Dominant Positive Effects on Both Constitutive and Ca²⁺-Triggered Vesicle Fusion in <i>syx^3–69</i> Heterozygotes

<div><p>(A) Models of multimeric SNARE complexes found in the wild type (+/+), the homozygous <i>syx^3–69</i> mutant <i>(syx/syx),</i> and the heterozygous <i>syx^3–69</i> mutant (<i>syx/</i>+). Oligomerization of a mixture of the wild-type and the mutant SNARE complex predicts that the T254I mutant syntaxin 1A has dominant positive effects on vesicle fusion. The wild-type syntaxin 1A is illustrated in red, whereas the T254I mutant syntaxin 1A is in blue. For simplicity, SNAP-25 is omitted from these models. PM, plasma membrane; SV, synaptic vesicle.</p> <p>(B–D) Representative traces of minis and evoked EPSPs in the heterozygous larvae are shown in (B) and (C), respectively. The average mini frequency and quantal content are shown in (D).</p> <p>(E) The histogram shows that the normalized mini frequency in the heterozygote is significantly higher than that in the wild type, but much lower than that in the homozygous mutant. ***, <i>p</i> < 0.001.</p> <p>(F) A histogram of the average EPSP amplitude recorded from the wild type (+/+), the heterozygote (<i>syx/</i>+), and the homozygote <i>(syx/syx)</i> at three different Ca²⁺ concentrations: 0.4 mM, 0.8 mM, and 1 mM. At these Ca²⁺ concentrations, the amplitude of EPSPs in the wild type is consistently lower than those in the heterozygote and the homozygote. Note that the difference between the wild type and mutants (the heterozygote and the homozygote) appears more dramatic at lower Ca²⁺ concentrations. At higher Ca²⁺ concentrations, this difference becomes smaller because EPSPs reach the “ceiling” set by the reversal potential. The amplitude of EPSPs is similar between the heterozygote and the homozygote. **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001.</p></div