Daren R. Ure

Mutant Huntingtin Impairs Axonal Trafficking in Mammalian Neurons In Vivo and In Vitro

ABSTRACT Recent data in invertebrates demonstrated that huntingtin (htt) is essential for fast axonal trafficking. Here, we provide direct and functional evidence that htt is involved in fast axonal trafficking in mammals. Moreover, expression of full-length mutant htt (mhtt) impairs vesicular and mitochondrial trafficking in mammalian neurons in vitro and in whole animals in vivo. Particularly, mitochondria become progressively immobilized and stop more frequently in neurons from transgenic animals. These defects occurred early in development prior to the onset of measurable neurological or mitochondrial abnormalities. Consistent with a progressive loss of function, wild-type htt, trafficking motors, and mitochondrial components were selectively sequestered by mhtt in human Huntingtons disease-affected brain. Data provide a model for how loss of htt function causes toxicity; mhtt-mediated aggregation sequesters htt and components of trafficking machinery leading to loss of mitochondrial motility and eventual mitochondrial dysfunction.

Naturally Occurring Human IgM Antibody That Binds B7-DC and Potentiates T Cell Stimulation by Dendritic Cells

A human IgM Ab, serum-derived human IgM 12 (sHIgM12), is identified that binds mouse and human dendritic cells (DC), inducing dramatic immunopotentiation following treatment of the mouse DC in vitro. Competition, transfection, and knockout studies identified the ligand on mouse DC as the costimulatory molecule family member B7-DC. Potent T cell responses are stimulated by Ag-pulsed DC treated with the sHIgM12 Ab in vitro and upon adoptive transfer of Ab-treated Ag-pulsed DC into animals. The multivalent structure of pentameric IgM provides the potential for cross-linking cell surface targets, endowing the soluble Abs with biological potential not normally associated with immune function. The ability of the sHIgM12 Ab to potentiate the immune response is dependent on the multimeric structure of IgM, as bivalent monomers do not retain this property. Furthermore, pretreatment of DC with IgM monomers blocks subsequent potentiation by intact IgM pentamers, an indication that cross-linking of B7-DC on the cell surface is critical for potentiation of Ag presentation. These findings imply that, in addition to known costimulatory roles, B7-DC can function as a receptor for signals delivered by cells expressing B7-DC ligands.

Polyreactive antibodies to glatiramer acetate promote myelin repair in murine model of demyelinating disease

Using a murine model of demyelinating disease, we demonstrate that remyelination of spinal cord axons is promoted by antibodies to glatiramer acetate (GA, Copolymer‐1, Copaxone®), a therapeutic agent for multiple sclerosis (MS). Glatiramer acetate is a mixture of randomly synthesized peptides that induces both T cell activation and antibody production in all treated individuals. These observations prompted us to compare the independent effects of adoptively transferred GA‐reactive T cells and antibodies in mice with chronic inflammatory demyelination induced by Theilers virus. Transferred T cells had no effect on lesion load or the extent of remyelination. Purified polyclonal GA antibodies also did not alter lesion load, which suggests that neither GA T cells or antibodies were pathogenic. On the contrary, GA antibodies enhanced the normally low level of remyelination in chronic lesions. The antibodies, which were primarily immunoglobulin (Ig) G1 and IgG2, cross‐reacted with oligodendrocytes, perivascular infiltrating cells, astrocytes, and neurons in spinal cord sections. In glial cultures they bound subsets of early lineage oligodendrocytes and microglia. Thus, several mechanisms may have contributed to the promotion of remyelination. These results support the hypothesis that the antibody response in GA‐treated patients is beneficial by facilitating repair of demyelinated lesions.

Quantitative Ultrastructural Analysis of a Single Spinal Cord Demyelinated Lesion Predicts Total Lesion Load, Axonal Loss, and Neurological Dysfunction in a Murine Model of Multiple Sclerosis

Infection of susceptible mice with Theilers murine encephalomyelitis virus results in neurological dysfunction from progressive central nervous system demyelination that is pathologically similar to the human disease, multiple sclerosis. We hypothesized that the development of neuropathology proceeds down a final common pathway that can be accurately quantified within a single spinal cord lesion. To test this hypothesis, we conducted quantitative ultrastructural analyses of individual demyelinated spinal cord lesions from chronically infected mice to determine whether pathological variables assessed within a single lesion accurately predicted global assessments of morphological and functional disease course. Within lesions we assessed by electron microscopy the frequencies of normally myelinated, remyelinated, and demyelinated axons, as well as degenerating axons and intra-axonal mitochondria. The frequency of medium and large remyelinated fibers within a single lesion served as a powerful indicator of axonal preservation and correlated with preserved neurological function. The number of degenerating axons and increased intra-axonal mitochondria also correlated strongly with global measures of disease course, such as total lesion load, spinal cord atrophy, and neurological function. This is the first study to demonstrate that functional severity of disease course is evident within a single demyelinated lesion analyzed morphometrically at the ultrastructural level.

Direct Comparison of Demyelinating Disease Induced by the Daniel's Strain and BeAn Strain of Theiler's Murine Encephalomyelitis Virus

We compared CNS disease following intracere‐bral injection of SJL mice with Daniels (DA) and BeAn 8386 (BeAn) strains of Theilers murine encephalomyelitis virus (TMEV). In tissue culture, DA was more virulent then BeAn. There was a higher incidence of demyelination in the spinal cords of SJL/J mice infected with DA as compared to BeAn. However, the extent of demyelination was similar between virus strains when comparing those mice that developed demyelination. Even though BeAn infection resulted in lower incidence of demyelination in the spinal cord, these mice showed significant brain disease similar to that observed with DA. There was approximately 100 times more virus specific RNA in the CNS of DA infected mice as compared to BeAn infected mice. This was reflected by more virus antigen positive cells (macrophages/microglia and oligodendrocytes) in the spinal cord white matter of DA infected mice as compared to BeAn. There was no difference in the brain infiltrating immune cells of DA or BeAn infected mice. However, BeAn infected mice showed higher titers of TMEV specific antibody. Functional deficits as measured by Rotarod were more severe in DA infected versus BeAn infected mice. These findings indicate that the diseases induced by DA or BeAn are distinct.

Neutralization of chemokines RANTES and MIG increases virus antigen expression and spinal cord pathology during Theiler's virus infection

Abstract The role of chemokines during some viral infections is unpredictable because the inflammatory response regulated by these molecules can have two, contrasting effects—viral immunity and immunopathologic injury to host tissues. Using Theilers virus infection of SJL mice as a model of this type of disease, we have investigated the roles of two chemokines—regulated on activation, normal T cell-expressed and secreted (RANTES) chemokine and monokine induced by IFN-γ (MIG)—by treating mice with antisera that block lymphocyte migration. Control, infected mice showed virus persistence, mild inflammation and a small degree of demyelination in the white matter of the spinal cord at 6 weeks post-infection. Treatment of mice with RANTES antiserum starting at 2 weeks post-infection increased both viral antigen expression and the severity of inflammatory demyelination at 6 weeks post-infection. MIG antiserum increased the spread of virus and the proportion of spinal cord white matter with demyelination. Overall, viral antigen levels correlated strongly with the extent of pathology. At the RNA level, high virus expression was associated with low IL-2 and high IL-10 levels, and RANTES antiserum decreased the IL-2/IL-10 ratio. Our results suggest that RANTES and MIG participate in an immune response that attempts to restrict viral expression while limiting immunopathology and that anti-chemokine treatment poses the risk of exacerbating both conditions in the long term.