No abstract

Objective: Neurodegeneration is thought to be the primary cause of neurological disability in multiple sclerosis (MS). Dysfunctional RNA-binding proteins (RBPs) including their mislocalization from nucleus to cytoplasm, stress granule formation, and altered RNA metabolism have been found to underlie neurodegeneration in amyotrophic lateral sclerosis and frontotemporal dementia. Yet, little is known about the role of dysfunctional RBPs in the pathogenesis of neurodegeneration in MS. As a follow-up to our seminal finding of altered RBP function in a single case of MS, we posited that there would be evidence of RBP dysfunction in cortical neurons in MS. Methods: Cortical neurons from 12 MS and six control cases were analyzed by immunohistochemistry for heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and TAR-DNA-binding protein-43 (TDP-43). Seven distinct neuronal phenotypes were identified based on the nucleocytoplasmic staining of these RBPs. Statistical analyses were performed by analyzing each phenotype in relation to MS versus controls. Results: Analyses revealed a continuum of hnRNP A1 and TDP-43 nucleocytoplasmic staining was found in cortical neurons, from neurons with entirely nuclear staining with little cytoplasmic staining in contrast to those with complete nuclear depletion of RBPs concurrent with robust cytoplasmic staining. The neuronal phenotypes that showed the most nucleocytoplasmic mislocalization of hnRNP A1 and TDP-43 statistically distinguished MS from control cases (P < 0.01, P < 0.001, respectively). Interpretation: The discovery of hnRNP A1 and TDP-43 nucleocytoplasmic mislocalization in neurons in MS brain demonstrate that dysfunctional RBPs may play a role in neurodegeneration in MS, as they do in other neurological diseases.

Neurodegeneration, including loss of neurons and axons, is a feature of progressive forms of multiple sclerosis (MS). The mechanisms underlying neurodegeneration are mostly unknown. Research implicates autoimmunity to nonmyelin self‐antigens as important contributors to disease pathogenesis. Data from our lab implicate autoimmunity to the RNA binding protein (RBP) heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a possible mechanism of neurodegeneration in MS. MS patients make antibodies to hnRNP A1, which have been shown to lead to neuronal dysfunction in vitro. Using an animal model of MS, experimental autoimmune encephalomyelitis (EAE), we show here that injection of anti‐hnRNP A1 antibodies, in contrast to control antibodies, resulted in worsened disease and increased neurodegeneration. We found a reduction of NeuN+ neuronal cell bodies in areas of the ventral gray matter of the spinal cord where anti‐hnRNP A1 antibodies localized. Neurons displayed increased levels of hnRNP A1 nucleocytoplasmic mislocalization and stress granule formation, both markers of neuronal injury. Anti‐hnRNP A1 antibodies were found to surround neuronal cell bodies and interact with CD68+ immune cells via Fc receptors. Additionally, anti‐hnRNP A1 antibodies were found within neuronal cell bodies including those of the ventral spinocerebellar tract (VSCT), a tract previously shown to undergo neurodegeneration in anti‐hnRNP A1 antibody injected EAE mice. Finally, both immune cells and neurons showed increased levels of inducible nitric oxide synthase, another indicator of cell damage. These findings suggest that autoimmunity to RBPs, such as hnRNP A1, play a role in neurodegeneration in EAE with important implications for the pathogenesis of MS.

Understanding the molecular mechanism of specific and polarized termination of DNA replication at a sequence-specific replication terminus requires detailed analyses of the interaction of terminator protein {ter) with specific DNA sequences (T], constituting the replication terminus. Such analyses should provide the structural basis of the functional polarity of replication inhibition observed in vivo and in vitro at T sites. With this objective in mind, we have purified the replication terminator protein of Escherichia coli to homogeneity and have analyzed the interaction of the protein with the replication termini of R6K, using chemical probes and by site-directed mutagenesis. The results show that one monomer of ter protein binds to a single T site with an equilibrium dissociation constant of 5 x 10"' moles/liter. Furthermore, a combination of alkylation interference and protection, hydroxyradical footprinting, and site-directed mutagenesis has revealed the phosphate groups and base residues of the T core sequence that make contacts with ter protein and those residues that are important for both DNA-protein interaction and for termination of replication in vivo. The overall picture that emerges from these analyses reveals that ter forms an asymmetric complex with a T sequence. Thus, the asymmetric ter-r complex provides a structural basis for the functional polarity of the arrest of a moving replication fork at a T site.