Cuprizone-induced demyelination triggers a CD8-pronounced T cell recruitment.

The loss of myelinating oligodendrocytes is a key characteristic of many neurological diseases, including Multiple Sclerosis (MS). In progressive MS, where effective treatment options are limited, peripheral immune cells can be found at the site of demyelination and are suggested to play a functional role during disease progression. In this study, we hypothesize that metabolic oligodendrocyte injury, caused by feeding the copper chelator cuprizone, is a potent trigger for peripheral immune cell recruitment into the central nervous system (CNS). We used immunohistochemistry and flow cytometry to evaluate the composition, density, and activation status of infiltrating T lymphocytes in cuprizone-intoxicated mice and post-mortem progressive MS tissues. Our results demonstrate a predominance of CD8+ T cells along with high proliferation rates and cytotoxic granule expression, indicating an antigenic and pro-inflammatory milieu in the CNS of cuprizone-intoxicated mice. Numbers of recruited T cells and the composition of lymphocytic infiltrates in cuprizone-intoxicated mice were found to be comparable to those found in progressive MS lesions. Finally, amelioration of the cuprizone-induced pathology by treating mice with laquinimod significantly reduces the number of recruited T cells. Overall, this study provides strong evidence that toxic demyelination is a sufficient trigger for T cells to infiltrate the demyelinated CNS. Further investigation of the mode of action and functional consequence of T cell recruitment might offer promising new therapeutic approaches for progressive MS.

Laquinimod ameliorates secondary brain inflammation.

Accumulating evidence suggests that a degenerative processes within the brain can trigger the formation of new, focal inflammatory lesions in Multiple Sclerosis (MS). Here, we used a novel pre-clinical MS animal model to test whether the amelioration of degenerative brain events reduces the secondary recruitment of peripheral immune cells and, in consequence, inflammatory lesion development. Neural degeneration was induced by a 3 weeks cuprizone intoxication period. To mitigate the cuprizone-induced pathology, animals were treated with Laquinimod (25 mg/kg) during the cuprizone-intoxication period. At the beginning of week 6, encephalitogenic T cell development in peripheral lymphoid organs was induced by the immunization with myelin oligodendrocyte glycoprotein 35-55 peptide (i.e., Cup/EAE). Demyelination, axonal injury and reactive gliosis were determined by immunohistochemistry. Positron emission tomography (PET) imaging was performed to analyze glia activation in vivo. Vehicle-treated cuprizone mice displayed extensive callosal demyelination, glia activation and enhanced TSPO-ligand binding. This cuprizone-induced pathology was profoundly ameliorated in mice treated with Laquinimod. In vehicle-treated Cup/EAE mice, the cuprizone-induced pathology triggered massive peripheral immune cell recruitment into the forebrain, evidenced by multifocal perivascular inflammation, glia activation and neuro-axonal injury. While anti myelin oligodendrocyte glycoprotein 35-55 peptide immune responses were comparable in vehicle- and Laquinimod-treated Cup/EAE mice, the cuprizone-triggered immune cell recruitment was ameliorated by the Laquinimod treatment. This study clearly illustrates that amelioration of a primary brain-intrinsic degenerative process secondary halts peripheral immune cell recruitment and, in consequence, inflammatory lesion development. These findings have important consequences for the interpretation of the results of clinical studies.

Expression of Translocator Protein and [18F]-GE180 Ligand Uptake in Multiple Sclerosis Animal Models.

Positron emission tomography (PET) ligands targeting the translocator protein (TSPO) represent promising tools to visualize neuroinflammation in multiple sclerosis (MS). Although it is known that TSPO is expressed in the outer mitochondria membrane, its cellular localization in the central nervous system under physiological and pathological conditions is not entirely clear. The purpose of this study was to assess the feasibility of utilizing PET imaging with the TSPO tracer, [18F]-GE180, to detect histopathological changes during experimental demyelination, and to determine which cell types express TSPO. C57BL/6 mice were fed with cuprizone for up to 5 weeks to induce demyelination. Groups of mice were investigated by [18F]-GE180 PET imaging at week 5. Recruitment of peripheral immune cells was triggered by combining cuprizone intoxication with MOG35⁻55 immunization (i.e., Cup/EAE). Immunofluorescence double-labelling and transgene mice were used to determine which cell types express TSPO. [18F]-GE180-PET reliably detected the cuprizone-induced pathology in various white and grey matter regions, including the corpus callosum, cortex, hippocampus, thalamus and caudoputamen. Cuprizone-induced demyelination was paralleled by an increase in TSPO expression, glia activation and axonal injury. Most of the microglia and around one-third of the astrocytes expressed TSPO. TSPO expression induction was more severe in the white matter corpus callosum compared to the grey matter cortex. Although mitochondria accumulate at sites of focal axonal injury, these mitochondria do not express TSPO. In Cup/EAE mice, both microglia and recruited monocytes contribute to the TSPO expressing cell populations. These findings support the notion that TSPO is a valuable marker for the in vivo visualization and quantification of neuropathological changes in the MS brain. The pathological substrate of an increase in TSPO-ligand binding might be diverse including microglia activation, peripheral monocyte recruitment, or astrocytosis, but not axonal injury.

Cuprizone-induced graded oligodendrocyte vulnerability is regulated by the transcription factor DNA damage-inducible transcript 3.

Oligodendrocytes are integral to efficient neuronal signaling. Loss of myelinating oligodendrocytes is a central feature of many neurological diseases, including multiple sclerosis (MS). The results of neuropathological studies suggest that oligodendrocytes react with differing sensitivity to toxic insults, with some cells dying early during lesion development and some cells being resistant for weeks. This proposed graded vulnerability has never been demonstrated but provides an attractive window for therapeutic interventions. Furthermore, the biochemical pathways associated with graded oligodendrocyte vulnerability have not been well explored. We used immunohistochemistry and serial block-face scanning electron microscopy (3D-SEM) to show that cuprizone-induced metabolic stress results in an "out of phase" degeneration of oligodendrocytes. Although expression induction of stress response transcription factors in oligodendrocytes occurs within days, subsequent oligodendrocyte apoptosis continues for weeks. In line with the idea of an out of phase degeneration of oligodendrocytes, detailed ultrastructural reconstructions of the axon-myelin unit demonstrate demyelination of single internodes. In parallel, genome wide array analyses revealed an active unfolded protein response early after initiation of the cuprizone intoxication. In addition to the cytoprotective pathways, the pro-apoptotic transcription factor DNA damage-inducible transcript 3 (DDIT3) was induced early in oligodendrocytes. In advanced lesions, DDIT3 was as well expressed by activated astrocytes. Toxin-induced oligodendrocyte apoptosis, demyelination, microgliosis, astrocytosis, and acute axonal damage were less intense in the Ddit3-null mutants. This study identifies DDIT3 as an important regulator of graded oligodendrocyte vulnerability in a MS animal model. Interference with this stress cascade might offer a promising therapeutic approach for demyelinating disorders.