Author Of 1 Presentation
YI01.03 - Inhibition of neuronal RNA binding protein dysfunction as a treatment for neurodegeneration in multiple sclerosis
Abstract
Background
Dysfunctional RNA binding proteins (RBPs), including heterogeneous nuclear ribonucleoprotein A1 (A1), are key pathological features of numerous neurologic diseases. Recently, evidence suggests that dysfunctional RBP biology, including mislocalization of the RBP from its homeostatic nuclear location to the cytoplasm, stress granule (SG) formation, and changes in RNA metabolism, are associated with neurodegeneration and worsened disease in multiple sclerosis (MS) and its models, such as experimental autoimmune encephalomyelitis (EAE).
Objectives
We hypothesized that correcting dysfunctional RBP biology with the nuclear export inhibitor, KPT-276, would restore A1 function by decreasing its nucleocytoplasmic mislocalization and reduce SG formation and neurodegeneration in spinal cord neurons from EAE animals as compared to vehicle controls.
Methods
C57BL/6 female mice were immunized with myelin oligodendrocyte glycoprotein (MOG35-55) to induce EAE. Upon reaching a clinical score of 2.5, mice were treated with KPT-276 (75 mg/kg, oral gavage) or vehicle control every other day for a total of six treatments. Two days following the final treatment, spinal cords were harvested and analyzed using immunofluorescence for A1 localization and SG formation in neurons as well as markers of neurodegeneration.
Results
EAE mice treated with KPT-276 showed a decrease in clinical score severity as compared to vehicle control EAE mice. Furthermore, mice treated with KPT-276 showed a significant reduction in the number of neurons with A1 mislocalization (*p=0.0351), the amount of A1 in the cytoplasm (*p=0.0341), and SG formation (*p=0.0208) as compared to vehicle mice. In the same areas of KPT-276 treated mice where there was a reduction in these features of dysfunctional RBP biology, there was a significant increase in the number of neurons indicative of increased neuronal survival (*p=0.0373).
Conclusions
These experiments suggest that modulating dysfunctional RBP biology through KPT-276 restores RBP function and is a potential therapeutic, which increases neuronal survival and decreases neurodegeneration and clinical disease severity.
Author Of 5 Presentations
P0425 - A real-world analysis of ocrelizumab treatment patterns among multiple sclerosis patients in Saskatchewan, Canada (ID 1443)
Abstract
Background
Ocrelizumab is a high-efficacy disease-modifying therapy (DMT) used for the treatment of multiple sclerosis (MS), which was recently added to the Saskatchewan (SK) formulary in May 2019. This medication has been approved for the treatment of relapsing-remitting multiple sclerosis (RRMS) and primary progressive multiple sclerosis (PPMS). However, there is limited data available on treatment patterns that could guide real-world clinical decision-making.
Objectives
To describe real-world evidence and treatment patterns of ocrelizumab in patients with multiple sclerosis in Saskatchewan, Canada.
Methods
We retrospectively collected data of research consented patients visiting the SK MS clinic who were ≥ 18 years of age with a confirmed diagnosis of MS and had received at least one treatment cycle of ocrelizumab. Data was extracted from our clinical electronic medical records and analyzed using SPSS.
Results
The study cohort consisted of 116 patients with the following demographics: mean age: 42.13 ± 10.5 years, 69 (59.5%) female, median expanded disability status scale (EDSS): 2.5 (0–6.5), and mean disease duration: 10.13 ± 7.7 years. The cohort received a median of 2 (1-5) ocrelizumab cycles at a mean duration of 9.22 ± 7.7 years from disease onset. Twenty two patients (19%) had PPMS, 92 patients (79.3%) had RRMS and 2 patients (1.7%) had active progressive MS. Fifty three patients (45.7%) were treatment naïve. The numbers of DMTs used in patients prior to switching to ocrelizumab were: one in 32 patients (27.6%); two in 16 patients (13.8%); three in 8 patients (6.9%); four in 5 patients (4.3%) and one patient (0.9%) each for five and seven DMTs. 22 patients (19%) switched from dimethyl fumarate; followed by 11 patients (9.5%) from glatiramer acetate, 9 patients (7.8%) from teriflunomide, 6 patients (5.2%) from interferon-beta 1a, 5 patients (4.3%) from natalizumab, 4 patients (3.4%) from alemtuzumab, 3 patients (2.6%) from fingolimod and 2 patients (1.7%) from interferon-beta 1b. The most common reasons for switching were adverse events and persistence of relapses. Infusion reactions were observed in 24 patients (20.7%) who started on ocrelizumab; with the most common presenting symptoms of fatigue, headache, nausea and throat irritation. Twenty four patients had a comorbid diagnosis of depression.
Conclusions
The majority of patients who started on ocrelizumab had RRMS. The most common reason to switch to ocrelizumab was due to adverse events or persistence of relapses.
P0934 - A novel experimental model to assess the contribution of heterogeneous nuclear ribonucleoprotein A1 (A1) mutations to neurodegeneration in MS. (ID 1500)
Abstract
Background
Current evidence indicates that neurodegeneration (NDG) is a prominent feature in the pathogenesis of MS, and the primary cause of disability in MS patients. Yet, knowledge of the molecular mechanisms of NDG in MS, as well as treatment options to prevent or reverse NDG, is lacking. Exploration of perturbed molecular mechanisms in MS may allow us to develop therapies that attenuate NDG and in turn, inhibit disability, and improve long-term quality of life of persons living with MS
Objectives
To characterize somatic (acquired) MS genetic mutations in A1 that cause molecular dysregulation of A1, using innovative cutting-edge optogenetic technology, and elucidate the role of dysregulated A1 in the pathogenesis of NDG in a model of MS.
Methods
We created optogenetic A1 protein expression constructs containing wildtype (WT) and mutant A1, tagged with both the optogene Cryptochrome 2 (Cry2) and mCherry. Cry2 is an optogenetic protein that self-clusters in response to blue light (BL) stimulation, and reverses when BL is turned off, therefore allowing real-time examination of protein clustering kinetics. We established an in vitro optogenetic paradigm of A1 dysfunction in HEK293T cells and gathered evidence of how select mutations affect A1 cellular localization and function.
Results
Using a chronic, single BL stimulus followed by a steady period of recovery (imitating chronic environmental cell stress), our data showed that the MS associated A1 mutations pP275S and pF281L increased the kinetics of both cytoplasmic cluster formation [17 and 32 minutes, respectively, compared to 51 minutes for WT (p < 0.0001)] and clearance of A1 [22 and 26 minutes, respectively, compared to 19 minutes for WT (p < 0.05)]. A1 clusters also decreased in quantity [clusters/cell: pF281L=2.1; pP275S=3.2; WT=3.4 (p < 0.05)] and increased in size [average cluster size (µm2): pF281L=0.37; pP275S=0.49; WT=0.24 (p < 0.001)], compared to WT. These data demonstrate changes in the molecular function of mutant A1 compared to WT, indicative of A1 dysfunction.
Conclusions
Using an in vitro optogenetic approach, this study presents evidence that somatic MS genetic mutations in A1, found in MS patient tissue, promotes A1 mislocalization and self-association, leading to protein dysfunction that may have an effect on NDG in MS pathogenesis.
P0937 - Altered expression of myelin-related RNA binding proteins in a mouse model of multiple sclerosis (ID 1782)
Abstract
Background
Abnormalities in expression of RNA-binding proteins (RBPs) have been shown to be involved in the pathogenesis of a number of disorders, most notably amyotrophic lateral sclerosis. Recent data demonstrates these RBP abnormalities were also found in the brains of patients with multiple sclerosis (MS) and MS models. Oligodendrocyte-mediated myelination of neuronal axons is essential for axonal integrity and protects neurons from degeneration. Myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) are the two major components of central nervous system (CNS) myelin. The transport and translation of MBP and MAG mRNAs in oligodendrocytes are regulated by diverse RBPs. Our lab previously demonstrated that dysfunction of RBPs including heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in neurons is a key contributor to neurodegenerative-mediated mechanisms in MS and its models. Therefore, neurodegeneration is a component of MS pathology, however, the underlying mechanisms behind this remain unknown.
Objectives
We tested the hypothesis that myelin-related RBPs are differentially expressed in the CNS of mice with experimental autoimmune encephalomyelitis (EAE).
Methods
EAE was induced in female C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG35-55). Animals (n=4 each EAE and naïve control) were observed clinically and spinal cord tissues were harvested at the peak of EAE for detection of the myelin related RBPs hnRNP A1, A2, K, E, and F using western blot.
Results
Quantitative analyses showed a significant increase in hnRNP A1 protein expression (p=0.007, unpaired tailed t-test) in spinal cords of mice with EAE. In contrast, hnRNP A2 (p=0.03) and hnRNP K (p=0.002) showed reduced expression. There were no significant differences in the expression levels of hnRNP E and F comparing EAE with naïve animals.
Conclusions
These data indicate that altered expression of the myelin-related RBPs hnRNP A1, hnRNP A2, and hnRNP K may contribute to demyelination and neurodegeneration in EAE, which might also apply to the pathogenesis of MS.
P0959 - Dysfunction of the RNA binding protein hnRNP A1 contributes to disease progression and neurodegeneration in an animal model of multiple sclerosis (ID 1437)
Abstract
Background
Loss of neurons and axons, collectively known as neurodegeneration, is characteristic of multiple sclerosis (MS), correlating with permanent disability. Dysfunctional RNA binding proteins (RBPs) underlie neurodegeneration in neurological diseases. Features of RBP dysfunction include mislocalization from its homeostatic nuclear location to the cytoplasm and formation of cytoplasmic stress granules (SGs). Neurons in MS brains display characteristic features of heterogenous nuclear ribonucleoprotein A1 (A1) dysfunction including its cytoplasmic mislocalization and depletion from the nucleus. Further, MS patients make antibodies to A1, and injections of anti-A1 antibodies into mice with experimental autoimmune encephalomyelitis (EAE – an animal model of MS) resulted in increased neurodegeneration.
Objectives
To determine how A1 dysfunction and anti-A1 antibodies contribute mechanistically to disease progression and NDG in an animal model of MS.
Methods
EAE was induced by immunization with myelin oligodendrocyte glycoprotein35-55. At symptom onset, mice were injected with anti-A1 antibodies or saline (control). Mice were sacrificed at five timepoints over a 21-day time course. Tissue was analyzed quantitatively for A1 mislocalization, SGs and neuronal loss (a marker of neurodegeneration) by immunohistochemistry.
Results
Mice in the EAE control group displayed neuronal A1 mislocalization (p<0.0001) and SG formation (p<0.01), which peaked at symptom onset concurrent with neuronal loss (p<0.01). After symptom onset, A1 mislocalization, SG formation returned to pre-symptomatic levels. Injections of anti-A1 antibodies into mice with EAE resulted in greater disability (p<0.05) and exacerbation of neuronal A1 mislocalization (p<0.05) and SG formation (p<0.05), including nuclear depletion of A1 (p<0.0001), a pathogenic neuronal phenotype in MS brains. These findings preceded neuronal cell loss. The anti-A1 antibodies also exacerbated neuronal cell loss (p<0.05), which did not recover and continued until the 21-day time point.
Conclusions
In contrast to controls, EAE mice injected with anti-A1 antibodies showed that RBP dysfunction occurred prior to neuronal cell body death (a marker of neurodegeneration) indicative of A1 dysfunction triggering, rather than resulting from neurodegeneration. The antibodies also exacerbated and caused permanent neuronal damage. These data reveal a novel mechanism of neurodegeneration, which can be targeted to inhibit disability in MS.
P0990 - Pro-inflammatory cytokines and autoantibodies induce dysfunctional RNA binding protein biology in primary cortical neurons (ID 1518)
Abstract
Background
Dysfunctional RNA binding proteins (RBPs), including heterogeneous nuclear ribonucleoprotein A1 (A1), have been suggested to play a role in neurodegeneration in multiple sclerosis (MS). Features of dysfunctional RBPs include a triad of molecular changes comprised of [i] stress granule (SG) formation, [ii] mislocalization of the RBP from its homeostatic nuclear location to the cytoplasm, and [iii] altered RNA metabolism. Previous studies have demonstrated that MS patients make antibodies to A1. The administration of anti-A1 antibodies to experimental autoimmune encephalomyelitis (EAE) mice exacerbated disease and induced the triad of molecular changes. Furthermore, in EAE mice, increased neuronal A1 mislocalization was observed in areas of the spinal cord with more inflammatory cytokine-producing T-cells. These data suggest a relationship between inflammation and dysfunctional RBPs.
Objectives
We hypothesized that the pro-inflammatory cytokines interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) and anti-A1 antibodies would induce dysfunctional RBP biology in primary cortical neurons.
Methods
Primary cortical neurons were isolated from C57BL/6 female mice. For cytokine experiments, neurons were treated for 24 hours with either IFNγ or TNFα at varying concentrations (0.625 μg/mL, 1.25 μg/mL, 2.5 μg/mL). For antibody experiments, neurons were treated for 24 hours with fluorescently conjugated IgG isotype control or anti-A1 antibodies at varying concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL). Following treatments, neurons were fixed and immunostained for endogenous A1 localization, SG formation, and beta III tubulin to assess changes in neurites (as a marker of neurodegeneration).
Results
Neurons treated with pro-inflammatory cytokines exhibited increased SG formation (****p<0.0001), increased A1 mislocalization (*p<0.0156), and decreased neurite length (****p<0.0001) compared to untreated controls. Neurons treated with anti-A1 antibodies also showed an increase in the number of neurons with SGs (*p<0.043), increased A1 mislocalization, and decreased neurite length (****p<0.0001) as compared to IgG and untreated controls.
Conclusions
These experiments suggest that pro-inflammatory cytokines and anti-A1 antibodies, both characteristics of the inflammatory response in MS, induce RBP dysfunction, including A1 mislocalization and SG formation, in primary cortical neurons, subsequently leading to neurodegeneration.