Claudia Wrzos

Assessment of lesion pathology in a new animal model of MS by multiparametric MRI and DTI

Magnetic resonance imaging (MRI) is the gold standard for the detection of multiple sclerosis (MS) lesions. However, current MRI techniques provide little information about the structural features of a brain lesion with inflammatory cell infiltration, demyelination, gliosis, acute axonal damage and axonal loss. To identify methods for a differentiation of demyelination, inflammation, and axonal damage we developed a novel mouse model combining cuprizone-induced demyelination and experimental autoimmune encephalomyelitis. MS-like brain lesions were assessed by T1-weighted, T2-weighted, and magnetization transfer MRI as well as by diffusion tensor imaging (DTI). T2-weighted MRI differentiated control and diseased mice, while T1-weighted MRI better reflected the extent of inflammation and axonal damage. In DTI, axonal damage and cellular infiltration led to a reduction of the axial diffusivity, whereas primary demyelination after cuprizone treatment was reflected by changes in radial but not axial diffusivity. Importantly, alterations in radial diffusivity were less pronounced in mice with demyelination, inflammation, and acute axonal damage, indicating that radial diffusivity may underestimate demyelination in acute MS lesions. In conclusion, the combined information from different DTI parameters allows for a more precise identification of solely demyelinated lesions versus demyelinated and acutely inflamed lesions. These findings are of relevance for offering individualized, stage-adapted therapies for MS patients.

Loss of Myelin Basic Protein Function Triggers Myelin Breakdown in Models of Demyelinating Diseases

Summary Breakdown of myelin sheaths is a pathological hallmark of several autoimmune diseases of the nervous system. We employed autoantibody-mediated animal models of demyelinating diseases, including a rat model of neuromyelitis optica (NMO), to target myelin and found that myelin lamellae are broken down into vesicular structures at the innermost region of the myelin sheath. We demonstrated that myelin basic proteins (MBP), which form a polymer in between the myelin membrane layers, are targeted in these models. Elevation of intracellular Ca2+ levels resulted in MBP network disassembly and myelin vesiculation. We propose that the aberrant phase transition of MBP molecules from their cohesive to soluble and non-adhesive state is a mechanism triggering myelin breakdown in NMO and possibly in other demyelinating diseases.

CD14 is a key organizer of microglial responses to CNS infection and injury.

Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll‐like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury‐associated factors. We show that its co‐receptor CD14 serves three non‐redundant functions in microglia. First, it confers an up to 100‐fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon β‐mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage‐associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo‐ or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non‐TLR systems to thereby fine‐tune microglial damage‐sensing capacity upon infectious and non‐infectious CNS challenges. GLIA 2016;64:635–649.

Acutely damaged axons are remyelinated in multiple sclerosis and experimental models of demyelination

Remyelination is in the center of new therapies for the treatment of multiple sclerosis to resolve and improve disease symptoms and protect axons from further damage. Although remyelination is considered beneficial in the long term, it is not known, whether this is also the case early in lesion formation. Additionally, the precise timing of acute axonal damage and remyelination has not been assessed so far. To shed light onto the interrelation between axons and the myelin sheath during de‐ and remyelination, we employed cuprizone‐ and focal lysolecithin‐induced demyelination and performed time course experiments assessing the evolution of early and late stage remyelination and axonal damage. We observed damaged axons with signs of remyelination after cuprizone diet cessation and lysolecithin injection. Similar observations were made in early multiple sclerosis lesions. To assess the correlation of remyelination and axonal damage in multiple sclerosis lesions, we took advantage of a cohort of patients with early and late stage remyelinated lesions and assessed the number of APP‐ and SMI32‐ positive damaged axons and the density of SMI31‐positive and silver impregnated preserved axons. Early de‐ and remyelinating lesions did not differ with respect to axonal density and axonal damage, but we observed a lower axonal density in late stage demyelinated multiple sclerosis lesions than in remyelinated multiple sclerosis lesions. Our findings suggest that remyelination may not only be protective over a long period of time, but may play an important role in the immediate axonal recuperation after a demyelinating insult.

Differential contribution of immune effector mechanisms to cortical demyelination in multiple sclerosis

Cortical demyelination is a widely recognized hallmark of multiple sclerosis (MS) and correlate of disease progression and cognitive decline. The pathomechanisms initiating and driving gray matter damage are only incompletely understood. Here, we determined the infiltrating leukocyte subpopulations in 26 cortical demyelinated lesions of biopsied MS patients and assessed their contribution to cortical lesion formation in a newly developed mouse model. We find that conformation-specific anti-myelin antibodies contribute to cortical demyelination even in the absence of the classical complement pathway. T cells and natural killer cells are relevant for intracortical type 2 but dispensable for subpial type 3 lesions, whereas CCR2+ monocytes are required for both. Depleting CCR2+ monocytes in marmoset monkeys with experimental autoimmune encephalomyelitis using a novel humanized CCR2 targeting antibody translates into significantly less cortical demyelination and disease severity. We conclude that biologics depleting CCR2+ monocytes might be attractive candidates for preventing cortical lesion formation and ameliorating disease progression in MS.

BCAS1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions

BCAS1 expression identifies newly formed and actively myelinating oligodendrocytes in development, adulthood, and disease. Mapping active myelination with BCAS1 Neuronal axon demyelination causes motor and cognitive impairments in multiple sclerosis (MS) and other demyelinating disorders. Although remyelinating strategies have been proposed, the lack of markers to detect areas of active myelination hampers the development of effective therapies. Fard et al. show that myelinating oligodendrocytes constitute a unique population expressing breast carcinoma amplified sequence 1 (BCAS1) in rodent and human brain tissue. In brain samples from deceased MS patients, BCAS1+ cells are present around lesions, suggesting that remyelination might occur during MS and that BCAS1 expression could be used to track responses to remyelinating compounds for treating demyelinating disorders. Investigations into brain function and disease depend on the precise classification of neural cell types. Cells of the oligodendrocyte lineage differ greatly in their morphology, but accurate identification has thus far only been possible for oligodendrocyte progenitor cells and mature oligodendrocytes in humans. We find that breast carcinoma amplified sequence 1 (BCAS1) expression identifies an oligodendroglial subpopulation in the mouse and human brain. These cells are newly formed, myelinating oligodendrocytes that segregate from oligodendrocyte progenitor cells and mature oligodendrocytes and mark regions of active myelin formation in development and in the adult. We find that BCAS1+ oligodendrocytes are restricted to the fetal and early postnatal human white matter but remain in the cortical gray matter until old age. BCAS1+ oligodendrocytes are reformed after experimental demyelination and found in a proportion of chronic white matter lesions of patients with multiple sclerosis (MS) even in a subset of patients with advanced disease. Our work identifies a means to map ongoing myelin formation in health and disease and presents a potential cellular target for remyelination therapies in MS.

A new mouse model of inflammatory cortical demyelination

Cortical damage is prominent in multiple sclerosis (MS) and associated with disease progression and cognitive impairment. Although subpial cortical demyelination is more specific for MS than demyelination of the white matter, little is known about the pathogenic requirements for inflammatory cortical demyelination. This study combines transgenic mice on a C57BL/6 background with a stereotactic targeting approach to characterize the immunological mechanisms of inflammatory cortical demyelination. Cortical demyelination as assessed by immunohistochemistry against myelin basic protein (MBP), was most pronounced at day 5 after lesion induction and paralleled by a significant reduction of p25+ adult and Olig2+ precursor oligodendrocytes. Time course experiments demonstrated the rapid resolution of cortical demyelination and a significant decrease of perivascular intracortical demyelination was already observable at day 20 after lesion induction. The number of APP+ axons was significantly higher around perivascularly demyelinated vessels, while the density of cortical neurons was not reduced. The inflammatory cortical infiltrate was composed of inflammatory monocytes, T cells, polymorphonuclear cells (PMNs) and natural killer (NK) cells. We depleted each cell population genetically or by antibody mediated cell depletion to assess their relevance for inflammatory cortical demyelination. A pathogenic antibody response and inflammatory monocytes were required for subpial cortical demyelination. Perivascular cortical demyelination was required in addition to encephalitogenic T cells and was augmented by NK cells. Adoptive transfer studies in RAG mice demonstrated that the antigen presenting functions of B cells were dispensable. In summary, we developed and characterized a novel inflammatory cortical demyelination model.

Early breakdown of the blood–brain barrier in a model of neuromyelitis optica

TY09 or HBPCT. As controls, conditionally-immortalized human microvascular endothelial cell line (FH-BNB) and pericyte cell line (HPPCT), both of which were derived from human blood–nerve barrier (BNB), were used in each of the experiments. Results: The conditioned media derived from HBPCT as well as TY09 up-regulated the expression of AQP4 at both of the mRNA and protein levels. The mRNA and protein of AQP4 in hAST also increased by the co-culture with TY09 and HBPCT, respectively. On the other hand, neither condition media nor co-cultures with FH-BNB as well as HPPCT did not affect the expression of AQP4 in hAST. Conclusions: Our results using a new human in vitro BBB models suggest that the humoral factor released from endothelial cells and pericytes composing the BBB regulates the expression level of AQP4 in astrocyte.