Cuprizone [bis(cyclohexylidenehydrazide)]-induced toxic demyelination is an experimental animal model commonly used to study de- and remyelination in the central nervous system. In this model, mice are fed with the copper chelator cuprizone which leads to oligodendrocyte death with subsequent demyelination. The underlying mechanisms of cuprizone-induced oligodendrocyte death are still unknown, and appropriate in vitro investigations to study these mechanisms are not available. Thus, we studied cuprizone effects on rat primary glial cell cultures and on the neuroblastoma cell line SH-SY5Y. Treatment of cells with different concentrations of cuprizone failed to show effects on the proliferation and survival of SH-SY5Y cells, microglia, astrocytes, and oligodendrocyte precursor cells (OPC). In contrast, differentiated mature oligodendrocytes (OL) were found to be significantly affected by cuprizone treatment. This was accompanied by a reduced mitochondrial potential in cuprizone-treated OL. These results demonstrate that the main toxic target for cuprizone is mature OL, whilst other glial cells including OPC are not or only marginally affected. This explains the selective demyelination induced by cuprizone in vivo.

No abstract

Administration of mesenchymal stromal cells (MSC) improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS) as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice), NSC-34 cells and glial cells (astrocytes, microglia) (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice) in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM) attenuated staurosporine (STS) - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF) in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1) was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS.

For the treatment of patients with multiple sclerosis there are no regenerative approaches to enhance remyelination. Mesenchymal stem cells (MSC) have been proposed to exert such regenerative functions. Intravenous administration of human MSC reduced the clinical severity of experimental autoimmune encephalomyelitis (EAE), an animal model mimicking some aspects of multiple sclerosis. However, it is not clear if this effect was achieved by systemic immunomodulation or if there is an active neuroregeneration in the central nervous system (CNS). In order to investigate remyelination and regeneration in the CNS we analysed the effects of intravenously and intranasally applied murine and human bone marrow-derived MSC on cuprizone induced demyelination, a toxic animal model which allows analysis of remyelination without the influence of the peripheral immune system. In contrast to EAE no effects of MSC on de- and remyelination and glial cell reactions were found. In addition, neither murine nor human MSC entered the lesions in the CNS in this toxic model. In conclusion, MSC are not directed into CNS lesions in the cuprizone model where the blood-brain-barrier is intact and thus cannot provide support for regenerative processes.

Summary2-Chlorodeoxyadenosine (cladribine, CdA) is an immunosuppressive drug that is licensed to treat hairy cell leukaemia, and has been shown recently to have beneficial effects in patients with multiple sclerosis (MS). The therapeutic effects of CdA have been suggested to be mediated partly through its potent toxicity towards lymphocytes. However, the effects of CdA on other immune cells are poorly understood. In the present study, we investigated the effects of CdA on the induction of apoptosis in human monocytes, monocyte-derived immature (ImDC) and mature (mDC) dendritic cells. Treatment of monocytes with CdA strongly induced apoptosis after 24 h, while apoptosis induction in DC was evident after 72 h. Furthermore, CdA treatment strongly induced caspase-3 and caspase-9 in monocytes, whereas activation of caspases was undetected in DC. The mitochondrial membrane potential in DC was reduced significantly after CdA treatment. DNA hypodiploid assessment showed fragmented nuclei in DC after CdA treatment together with activation of p53 protein. These results revealed that CdA induces caspase-independent apoptosis in DC and suggest cell type specific effects of CdA. This mechanism may contribute to the effect of CdA in autoimmune diseases.