Moses Rodriguez

Efficient central nervous system remyelination requires T cells

We demonstrate a role for immune functions in the spontaneous remyelination of central nervous system (CNS) axons after lysolecithin‐induced demyelination in the spinal cord. Rag‐1–deficient mice lack both B cells and T cells and show significantly reduced spontaneous remyelination compared with control mice of matching genetic background. Mice lacking or depleted of either CD4+ T cells or CD8+ T cells also exhibit reduced remyelination. These data indicate that T cells are necessary for efficient CNS remyelination. Thus, general nonspecific immunosuppression as a therapeutic approach for the treatment of CNS injury and demyelinating disease may have undesirable effects on subsequent tissue repair. Ann Neurol 2003;53:680–684

Human antibodies accelerate the rate of remyelination following lysolecithin-induced demyelination in mice

Immunoglobulin‐based therapies are becoming increasingly common for the treatment of neurologic and autoimmune diseases in humans. In this study, we demonstrate that systemic administration of either polyclonal human immunoglobulins or specific human monoclonal antibodies can accelerate the rate of CNS remyelination following toxin‐induced demyelination. Injection of lysolecithin directly into the spinal cord results in focal demyelinated lesions. In contrast to other murine models of demyelinating disease, the mechanism of demyelination following lysolecithin injection is independent of immune system activation, and chronic inflammation at the site of the lesion is minimal. Administration of polyclonal human IgM (pHIgM) or a serum‐derived human monoclonal antibody (sHIgM22) resulted in approximately a twofold increase in remyelinating axons when compared to animals treated with saline or with antibodies that do not promote repair. Both pHIgM and sHIgM22 show strong binding to CNS white matter and oligodendrocytes, while antibodies that did not accelerate remyelination do not. This differential staining pattern suggests that enhanced remyelination may result from direct stimulation of oligodendrocyte remyelination by binding to surface receptors on oligodendrocytes or glial progenitor cells. We propose the use of human polyclonal IgM or specific human monoclonal IgM antibodies as potential therapies to enhance myelin repair following CNS injury and disease. GLIA 37:241–249, 2002.

Targeting kallikrein 6 proteolysis attenuates CNS inflammatory disease

Kallikrein 6 (K6, MSP) is a newly identified member of the Kallikrein family of serine proteases that is preferentially expressed in the adult central nervous system (CNS). We have previously demonstrated that K6 is abundantly expressed by inflammatory cells at sites of CNS inflammation and demyelination in animal models of multiple sclerosis (MS) and in human MS lesions. To test the hypothesis that this novel enzyme is a mediator of pathogenesis in CNS inflammatory disease, we have evaluated whether autonomously generated K6 antibodies alter the clinicopathological course of disease in murine proteolipid protein139‐151‐induced experimental autoimmune encephalomyelitis (PLP139‐151 EAE). We demonstrate that immunization of mice with recombinant K6 generates antibodies that block K6 enzymatic activity in vitro, including the breakdown of myelin basic protein (MBP), and that K6‐ immunized mice exhibit significantly delayed onset and severity of clinical deficits. Reduced clinical deficits were reflected in significantly less spinal cord pathology and meningeal inflammation and in reduced Th1 cellular responses in vivo and in vitro. These data demonstrate for the first time that K6 participates in enzymatic cascades mediating CNS inflammatory disease and that this unique enzyme may represent a novel therapeutic target for the treatment of progressive inflammatory disorders, including MS.

Remyelination-promoting antibodies activate distinct Ca2+ influx pathways in astrocytes and oligodendrocytes: relationship to the mechanism of myelin repair

Our laboratory has identified mouse and human monoclonal antibodies that promote myelin repair in multiple models of demyelinating disease. We have proposed that these antibodies promote remyelination by directly activating central nervous system glia. Intracellular calcium concentration was monitored using a Fura2 ratiometric assay. Repair-promoting antibodies induced distinct Ca2+ signals in both astrocytes and oligodendrocytes. Astrocyte Ca2+ signaling is mediated by a phospholipase C-dependent pathway while oligodendrocyte Ca2+ signaling is mediated via AMPA-sensitive glutamate receptors. An antibodys ability to induce Ca2+ signals is statistically correlated with promotion of myelin repair. These findings support the hypothesis that remyelination-promoting antibodies are acting directly at the surface of glial cells to induce calcium-dependent physiologic reparative function.

MSP, a trypsin-like serine protease, is abundantly expressed in the human nervous system

The goal of the present investigation was to determine the regional and cellular specific expression patterns of the newly identified serine protease, myelencephalon‐specific protease (MSP), in the adult human brain (Scarisbrick et al. [ 1997b ] J. Neurosci. 17:8156–8168). To assess the potential scope of MSP activity, Northern blot techniques were used to determine the relative abundance of MSP mRNA in 16 different adult human brain regions, and in the brain and peripheral tissues of the midgestation human fetus. The regional and temporal specific expression patterns of MSP mRNA were directly compared with those of tissue plasminogen activator (tPA), a serine protease strongly implicated in the development, ongoing plasticity, and response of the nervous system to injury and disease. mRNA encoding each protease was distributed widely throughout the normal adult human central nervous system (CNS), but the expression of each was only partially overlapping. Additionally, compared with tPA, MSP exhibited a more restricted distribution and delayed developmental onset. By immunohistochemical localization, MSP was present at moderate to high levels in neurons and oligodendroglia of the adult human brain, at a level closely resembling the relative abundance indicated by Northern blot. MSP was most abundantly expressed in the spinal cord, hippocampus, substantia nigra, and basal ganglia. The robust expression of MSP in clinically significant regions of the adult human CNS indicates that further study of this protease in terms of both normal brain physiology and neurodegenerative disorders is warranted. J. Comp. Neurol. 431:347–361, 2001.

Distinct promoters regulate tissue‐specific and differential expression of kallikrein 6 in CNS demyelinating disease

Kallikrein 6 is a serine protease expressed abundantly in normal adult human and rodent CNS, and therein is regulated by injury. In the case of CNS demyelinating disease, K6 expression in CNS occurs additionally in perivascular and parenchymal inflammatory cells suggesting a role in pathogenesis. Herein we describe two unique transcripts that occur within the human and mouse K6 genes that differ in their 5′‐untranslated regions. These transcripts have identical translation initiation sites in exon 3, are expressed in a tissue‐specific fashion and are differentially regulated in response to CNS injury. While the human and mouse 5′‐transcripts differ in sequence they are identical in genomic organization and tissue‐specific expression. The most 5′‐transcript, designated transcript 1, includes exon 1–7, and was detectable in all CNS regions, but not in any non‐CNS tissues examined (spleen, thymus, liver, kidney, pancreas, submandibular gland and peripheral nerve). In contrast, transcript 2 lacks exon 1, but contains a unique sequence at the 5′‐end of exon 2, designated exon 2A. Transcript 2 was expressed both in CNS and in each peripheral tissue. In a murine model of human CNS demyelinating inflammatory disease induced by Theilers picornovirus, mouse K6 transcript 1 was up‐regulated in brain and spinal cord at acute and more chronic phases of CNS inflammation and demyelination, while overall transcript 2 expression was not significantly altered. However, in isolated splenocyte cultures, transcript 2 was up‐regulated two‐fold by cellular activation. Tissue‐specific expression patterns and differential regulation in CNS disease indicates that each K6 5′‐transcript is probably regulated by unique promoter elements and may serve as a molecular target to treat inflammatory demyelinating disease.

Preferential expression of myelencephalon-specific protease by oligodendrocytes of the adult rat spinal cord white matter

Myelencephalon‐specific protease (MSP) is a novel serine protease that is expressed predominantly in the nervous system. In the adult rat spinal cord, MSP mRNA expression was dramatically upregulated, in both the white and gray matter, after systemic exposure to the glutamate receptor agonist, kainic acid (KA) (Scarisbrick et al. J Neurosci 17: 8156–8168, 1997b). To determine the cell‐specific expression patterns of MSP, we generated MSP‐specific monoclonal antibodies. These have been used in immunohistochemical and in situ hybridization colocalization studies, to demonstrate that MSP mRNA and protein are produced predominantly by CNP‐immunoreactive oligodendroglia, but not by GFAP‐immunoreactive astrocytes, in the white matter of the normal adult cord. In vitro, the soma of oligodendrocytes were also densely MSP immunoreactive, as were their growth tips, while astrocytes were associated with lower levels. These findings suggest that the enzymatic activity of MSP is likely to be important in the biology of oligodendrocytes and/or in the maintenance of the nerve fiber tracts of the adult spinal cord. GLIA 30:219–230, 2000.

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.

Kallikrein 6 regulates early CNS demyelination in a viral model of multiple sclerosis

Kallikrein 6 (Klk6) is a secreted serine protease that is elevated in active multiple sclerosis lesions and patient sera. To further evaluate the involvement of Klk6 in chronic progressive demyelinating disease, we determined its expression in the brain and spinal cord of SJL mice infected with Theilers murine encephalomyelitis virus (TMEV) and assessed the effects of Klk6‐neutralizing antibodies on disease progression. Klk6 RNA expression was elevated in the brain and spinal cord by 7 days postinfection (dpi). Thereafter, Klk6 expression persisted primarily in the spinal cord reaching a peak of fivefold over controls at mid‐chronic stages (60u2003dpi–120u2003dpi). Significant elevations in Klk6 RNA were also induced in splenocytes stimulated with viral capsid proteins in vitro and in activated human acute monocytic leukemia cells. Klk6‐neutralizing antibodies reduced TMEV‐driven brain and spinal cord pathology and delayed‐type hypersensitivity (DTH) responses when examined at early chronic time points (40u2003dpi). Reductions in spinal cord pathology included a decrease in activated monocytes/microglia and reductions in the loss of myelin basic protein (MBP). By 180u2003dpi, pathology scores no longer differed between groups. These findings point to regulatory activities for Klk6 in the development and progression of central nervous system (CNS) inflammation and demyelination that can be effectively targeted through the early chronic stages with neutralizing antibody.

Heterogeneity of Pathogenesis in Multiple Sclerosis: Implications for Promotion of Remyelination

Enhancing myelin repair remains an important therapeutic goal in primary demyelinating diseases of the central nervous system (CNS) such as multiple sclerosis (MS). The emerging heterogeneity of pathology within MS lesions, and differential oligodendrocyte survival in particular, suggests that therapeutic strategies may need to be tailored to an individual patients requirements. A number of therapeutic strategies have been proposed to enhance myelin repair in the CNS: cell transplantation, growth factor therapy, and antibody therapy, but each proposed therapy has different implications with respect to pathogenetic mechanisms of demyelination. Of these, antibody therapy is the most amenable to immediate application in patients-but a combination of therapeutic approaches may be required in practice.