Autoantibodies to heterogeneous nuclear ribonuclear protein A1 (hnRNPA1) cause altered 'ribostasis' and neurodegeneration; the legacy of HAM/TSP as a model of progressive multiple sclerosis.

Several years following its discovery in 1980, infection with human T-lymphotropic virus type 1 (HTLV-1) was shown to cause HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease biologically similar to progressive forms of multiple sclerosis (MS). In this manuscript, we review some of the clinical, pathological, and immunological similarities between HAM/TSP and MS with an emphasis on how autoantibodies to an RNA binding protein, heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), might contribute to neurodegeneration in immune mediated diseases of the central nervous system.

Antibodies to the RNA Binding Protein Heterogeneous Nuclear Ribonucleoprotein A1 Colocalize to Stress Granules Resulting in Altered RNA and Protein Levels in a Model of Neurodegeneration in Multiple Sclerosis.

OBJECTIVE: Multiple sclerosis (MS) is the most common demyelinating disorder of the central nervous system (CNS). Data suggest that antibodies to CNS targets contribute to the pathogenesis of MS. MS patients produce autoantibodies to heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). hnRNP A1 is an RNA binding protein (RBP) overexpressed in neurons that functions in pre-mRNA splicing, mRNA trafficking, and translation. Previously, we showed that anti-hnRNP A1 antibodies entered neuronal cells (in vitro) via clathrin-mediated endocytosis, caused mislocalization of endogenous hnRNP A1 protein and increased markers of neurodegeneration including decreased ATP concentration and apoptosis. In this study, we hypothesized that anti-hnRNP A1 antibodies might cause stress granule formation and altered levels of RNAs and proteins that bind hnRNP A1. METHODS: Neuronal cell lines were exposed to anti-hnRNP A1 and isotype-matched control antibodies in vitro and examined for neuronal granule formation, including stress granules, P bodies and transport granules. In addition, RNAs that bound hnRNP A1 were determined. Levels of RNA and their translated proteins were measured upon exposure to the anti-hnRNP A1 antibodies. RESULTS: Anti-hnRNP A1 antibodies induced and localized to stress granules, a marker of neurodegeneration, within a neuronal cell line. The anti-hnRNP A1 antibodies did not induce P bodies or neuronal granules. Clinically relevant RNAs were found to bind hnRNP A1. In addition, the anti-hnRNP A1 antibodies caused reduced levels of RNA and protein of the spinal paraplegia genes (SPGs) 4 and 7, which when mutated mimic progressive MS. CONCLUSIONS: Taken together, these data suggest potential mechanisms by which autoantibodies may contribute to neurodegeneration in MS.

Antibodies to the RNA-binding protein hnRNP A1 contribute to neurodegeneration in a model of central nervous system autoimmune inflammatory disease.

BACKGROUND: Neurodegeneration is believed to be the primary cause of permanent, long-term disability in patients with multiple sclerosis. The cause of neurodegeneration in multiple sclerosis appears to be multifactorial. One mechanism that has been implicated in the pathogenesis of neurodegeneration in multiple sclerosis is the targeting of neuronal and axonal antigens by autoantibodies. Multiple sclerosis patients develop antibodies to the RNA-binding protein, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which is enriched in neurons. We hypothesized that anti-hnRNP A1 antibodies would contribute to neurodegeneration in an animal model of multiple sclerosis. METHODS: Following induction of experimental autoimmune encephalomyelitis (EAE) by direct immunization with myelin oligodendrocyte glycoprotein, mice were injected with anti-hnRNP A1 or control antibodies. Animals were examined clinically, and the central nervous system (CNS) tissues were tested for neurodegeneration with Fluoro-Jade C, a marker of degenerating neural elements. RESULTS: Injection of anti-hnRNP A1 antibodies in mice with EAE worsened clinical disease, altered the clinical disease phenotype, and caused neurodegeneration preferentially in the ventral spinocerebellar tract and deep white matter of the cerebellum in the CNS. Neurodegeneration in mice injected with hnRNP A1-M9 antibodies compared to control groups was consistent with "dying back" axonal degeneration. CONCLUSIONS: These data suggest that antibodies to the RNA-binding protein hnRNP A1 contribute to neurodegeneration in immune-mediated disease of the CNS.

Neurodegeneration in multiple sclerosis involves multiple pathogenic mechanisms.

Multiple sclerosis (MS) is a complex autoimmune disease that impairs the central nervous system (CNS). The neurological disability and clinical course of the disease is highly variable and unpredictable from one patient to another. The cause of MS is still unknown, but it is thought to occur in genetically susceptible individuals who develop disease due to a nongenetic trigger, such as altered metabolism, a virus, or other environmental factors. MS patients develop progressive, irreversible, neurological disability associated with neuronal and axonal damage, collectively known as neurodegeneration. Neurodegeneration was traditionally considered as a secondary phenomenon to inflammation and demyelination. However, recent data indicate that neurodegeneration develops along with inflammation and demyelination. Thus, MS is increasingly recognized as a neurodegenerative disease triggered by an inflammatory attack of the CNS. While both inflammation and demyelination are well described and understood cellular processes, neurodegeneration might be defined by a diverse pool of any of the following: neuronal cell death, apoptosis, necrosis, and virtual hypoxia. In this review, we present multiple theories and supporting evidence that identify common biological processes that contribute to neurodegeneration in MS.

Autoantibodies to Non-myelin Antigens as Contributors to the Pathogenesis of Multiple Sclerosis.

For years, investigators have sought to prove that myelin antigens are the primary targets of autoimmunity in multiple sclerosis (MS). Recent experiments have begun to challenge this assumption, particularly when studying the neurodegenerative phase of MS. T-lymphocyte responses to myelin antigens have been extensively studied, and are likely early contributors to the pathogenesis of MS. Antibodies to myelin antigens have a much more inconstant association with the pathogenesis of MS. Recent studies indicate that antibodies to non-myelin antigens such as neurofilaments, neurofascin, RNA binding proteins and potassium channels may contribute to the pathogenesis of MS. The purpose of this review is to analyze recent studies that examine the role that autoantibodies to non-myelin antigens might play in the pathogenesis of MS.

Antibody transfection into neurons as a tool to study disease pathogenesis.

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP). How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1). Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis. Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions. In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

Pathogenic mechanisms of neurodegeneration based on the phenotypic expression of progressive forms of immune-mediated neurologic disease.

Considering there are no treatments for progressive forms of multiple sclerosis (MS), a comprehensive understanding of the role of neurodegeneration in the pathogenesis of MS should lead to novel therapeutic strategies to treat it. Many studies have implicated viral triggers as a cause of MS, yet no single virus has been exclusively shown to cause MS. Given this, human and animal viral models of MS are used to study its pathogenesis. One example is human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Importantly, HAM/TSP is similar clinically, pathologically, and immunologically to progressive MS. Interestingly, both MS and HAM/TSP patients were found to make antibodies to heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an RNA-binding protein overexpressed in neurons. Anti-hnRNP A1 antibodies reduced neuronal firing and caused neurodegeneration in neuronal cell lines, suggesting the autoantibodies are pathogenic. Further, microarray analyses of neurons exposed to anti-hnRNP A1 antibodies revealed novel pathways of neurodegeneration related to alterations of RNA levels of the spinal paraplegia genes (SPGs). Mutations in SPGs cause hereditary spastic paraparesis, genetic disorders clinically indistinguishable from progressive MS and HAM/TSP. Thus, there is a strong association between involvement of SPGs in neurodegeneration and the clinical phenotype of progressive MS and HAM/TSP patients, who commonly develop spastic paraparesis. Taken together, these data begin to clarify mechanisms of neurodegeneration related to the clinical presentation of patients with chronic immune-mediated neurological disease of the central nervous system, which will give insights into the design of novel therapies to treat these neurological diseases.