Astroglial and therapeutic factors affect demyelination in murine models with toxic demyelination

Astrocytes might play an important role in demyelinating diseases such as multiple sclerosis (MS) since serum autoantibodies directed against the astrocytic water channel aquaporin-4 cause inflammatory demyelinating lesions in the MS-related disease neuromyelitis optica. Oral cuprizone challenge leads to demyelination of the corpus callosum and cortex in mice, whereby the blood-brain-barrier remains intact. Astrocytic activation of nuclear factor kappa of activated B cells (NF-κB) plays a key role for mediating demyelination under cuprizone. Recent clinical trials in MS indicate that laquinimod (LAQ) is an oral substance with more pronounced effects on disability and brain atrophy than on relapses suggesting that LAQ might exert effects not only on peripheral immune cells, but also on central nervous system (CNS)-resident cells. The aim of the present study was to investigate intrinsic and therapy-induced astrocytic effects on cuprizone-induced pathology in mice. One aim was to assess the effects of LAQ on toxic demyelination. A further aim was to study the impact of increased astrocytic glial fibrillary acidic protein (GFAP) expression on cuprizone-induced changes. Eight- to ten-weeks-old male C57BL/6 mice were given 0.25% cuprizone for one or six weeks. Histological and immunohistochemical analyses were performed to evaluate therapy-induced and astrocytic effects on cuprizone-induced changes on glia, myelin and axons in the corpus callosum. In addition, astrocytic NF-κB activation was assessed by nuclear translocation of p65 in GFAP-positive astrocytes. To examine the effects of LAQ, mice were treated with 0, 5 or 25 mg/kg LAQ per day during cuprizone challenge. After one week of cuprizone, oligodendrocyte apoptosis was reduced by 62% in mice treated with 25 mg/kg LAQ compared to vehicle-treated animals. After six weeks of cuprizone, LAQ reduced demyelination in a dose-dependent manner and attenuated microglial activation, axonal damage and reactive astrogliosis compared to the vehicle group. Similar results were observed in recombination activating gene 1 (Rag1)-deficient mice constitutively lacking T and B cells indicating that the effect of LAQ in the cuprizone model is CNS-intrinsic. Astrocytic NF-κB activation was significantly decreased by 46% under 25 mg/kg LAQ compared to the vehicle group after six weeks of cuprizone. These data indicate that LAQ might protect from cuprizone-induced pathology through CNS-intrinsic mechanisms by reducing NF-κB activation in astrocytes. Recently published data in primary astrocytic cultures support these findings by showing that LAQ directly inhibited the astrocytic NF-κB activation and thereby down-regulated the astrocytic pro-inflammatory response. The impact of increased astrogliosis on cuprizone-induced demyelination was evaluated in transgenic mice overexpressing human GFAP. These animals show an increased astrogliosis even without external stimuli. After one week of cuprizone, transgenic mice displayed still higher densities of preserved mature oligodendrocytes and an 80% reduction of oligodendroglial apoptoses compared to the corresponding wild type animals. After six weeks of cuprizone, transgenic mice showed decreased demyelination, microglial activation and axonal damage as well as an 80% reduction of astrocytic NF-κB activation compared to wild type animals. These data indicate that reduced astrocytic NF-κB activation might also contribute to reduced cuprizone-induced pathology in mice overexpressing human GFAP. These data suggest that down-regulating the astrocytic NF-κB activation might be a potential therapeutic approach for the future treatment of demyelinating diseases such as MS. The CNS-intrinsic effects of LAQ on astrocytic activation might explain the clinical findings of more pronounced effects on disability and brain atrophy than on relapses. The findings from this work could contribute to a better understanding and further development of novel protective therapies limiting tissue damage in demyelinating diseases such as MS.