Investigating demyelination, efficient remyelination and remyelination failure in organotypic cerebellar slice cultures: Workflow and practical tips.

Healthy myelin is essential for proper brain function. When the myelin sheath is damaged, fast saltatory impulse conduction is lost and neuronal axons become vulnerable to degeneration. Thus, regeneration of the myelin sheath by encouraging oligodendrocyte lineage cells to remyelinate the denuded axons is a promising therapeutic target for demyelinating diseases such as multiple sclerosis. Ex vivo organotypic cerebellar slice cultures are a useful model to study developmental myelination, demyelination, remyelination and remyelination failure. In these cultures, the cerebellum's three-dimensional architecture and various cell populations remain largely intact, providing a realistic and relatively cost-efficient model that can be easily manipulated by the addition of viral vectors, pharmaceuticals or (transgenic) cells to augment or replace resident cell populations. Moreover, slice cultures can be treated with lysolecithin or polyinosinic:polycytidylic acid to induce demyelination and mimic efficient as well as inefficient remyelination. It can be challenging to set up slice cultures for the first time, as in our experience, seemingly minor differences in technique and materials can make a great difference to the quality of the cultures. Therefore, this report provides an in-depth description for the generation and maintenance of ex vivo organotypic cerebellar cultures for demyelination-remyelination studies with a focus on practical tips for scientists that are new to this technique.

Matrix metalloproteinases shape the oligodendrocyte (niche) during development and upon demyelination.

The oligodendrocyte lineage cell is crucial to proper brain function. During central nervous system development, oligodendrocyte progenitor cells (OPCs) migrate and proliferate to populate the entire brain and spinal cord, and subsequently differentiate into mature oligodendrocytes that wrap neuronal axons in an insulating myelin layer. When damage occurs to the myelin sheath, OPCs are activated and recruited to the demyelinated site, where they differentiate into oligodendrocytes that remyelinate the denuded axons. The process of OPC attraction and differentiation is influenced by a multitude of factors from the cell's niche. Matrix metalloproteinases (MMPs) are powerful and versatile enzymes that do not only degrade extracellular matrix proteins, but also cleave cell surface receptors, growth factors, signaling molecules, proteases and other precursor proteins, leading to their activation or degradation. MMPs are markedly upregulated during brain development and upon demyelinating injury, where their broad functions influence the behavior of neural progenitor cells (NPCs), OPCs and oligodendrocytes. In this review, we focus on the role of MMPs in (re)myelination. We will start out in the developing brain with describing the effects of MMPs on NPCs, OPCs and eventually oligodendrocytes. Then, we will outline their functions in oligodendrocyte process extension and developmental myelination. Finally, we will review their potential role in demyelination, describe their significance in remyelination and discuss the evidence for a role of MMPs in remyelination failure, focusing on multiple sclerosis. In conclusion, MMPs shape the oligodendrocyte (niche) both during development and upon demyelination, and thus are important players in directing the fate and behavior of oligodendrocyte lineage cells throughout their life cycle.

A quantitative neuropathological assessment of translocator protein expression in multiple sclerosis.

The 18 kDa translocator protein (TSPO) is increasingly used to study brain and spinal cord inflammation in degenerative diseases of the CNS such as multiple sclerosis. The enhanced TSPO PET signal that arises during disease is widely considered to reflect activated pathogenic microglia, although quantitative neuropathological data to support this interpretation have not been available. With the increasing interest in the role of chronic microglial activation in multiple sclerosis, characterising the cellular neuropathology associated with TSPO expression is of clear importance for understanding the cellular and pathological processes on which TSPO PET imaging is reporting. Here we have studied the cellular expression of TSPO and specific binding of two TSPO targeting radioligands (3H-PK11195 and 3H-PBR28) in tissue sections from 42 multiple sclerosis cases and 12 age-matched controls. Markers of homeostatic and reactive microglia, astrocytes, and lymphocytes were used to investigate the phenotypes of cells expressing TSPO. There was an approximate 20-fold increase in cells double positive for TSPO and HLA-DR in active lesions and in the rim of chronic active lesion, relative to normal appearing white matter. TSPO was uniformly expressed across myeloid cells irrespective of their phenotype, rather than being preferentially associated with pro-inflammatory microglia or macrophages. TSPO+ astrocytes were increased up to 7-fold compared to normal-appearing white matter across all lesion subtypes and accounted for 25% of the TSPO+ cells in these lesions. To relate TSPO protein expression to ligand binding, specific binding of the TSPO ligands 3H-PK11195 and 3H-PBR28 was determined in the same lesions. TSPO radioligand binding was increased up to seven times for 3H-PBR28 and up to two times for 3H-PK11195 in active lesions and the centre of chronic active lesions and a strong correlation was found between the radioligand binding signal for both tracers and the number of TSPO+ cells across all of the tissues examined. In summary, in multiple sclerosis, TSPO expression arises from microglia of different phenotypes, rather than being restricted to microglia which express classical pro-inflammatory markers. While the majority of cells expressing TSPO in active lesions or chronic active rims are microglia/macrophages, our findings also emphasize the significant contribution of activated astrocytes, as well as smaller contributions from endothelial cells. These observations establish a quantitative framework for interpretation of TSPO in multiple sclerosis and highlight the need for neuropathological characterization of TSPO expression for the interpretation of TSPO PET in other neurodegenerative disorders.

MMP7 cleaves remyelination-impairing fibronectin aggregates and its expression is reduced in chronic multiple sclerosis lesions.

Upon demyelination, transient expression of fibronectin precedes successful remyelination. However, in chronic demyelination observed in multiple sclerosis (MS), aggregates of fibronectin persist and contribute to remyelination failure. Accordingly, removing fibronectin (aggregates) would constitute an effective strategy for promoting remyelination. Matrix metalloproteinases (MMPs) are enzymes known to remodel extracellular matrix components, including fibronectin. Here, we examined the ability of MMPs to degrade fibronectin aggregates. Our findings reveal that MMP7 cleaved fibronectin aggregates resulting into a prominent 13 kDa EIIIA (16 kDa EDA)-containing fragment. MMP7 was upregulated during lysolecithin-induced demyelination, indicating its potential for endogenous fibronectin clearance. In contrast, the expression of proMMP7 was substantially decreased in chronic active and inactive MS lesions compared with control white matter and remyelinated MS lesions. Microglia and macrophages were major cellular sources of proMMP7 and IL-4-activated, but not IFNγ+LPS-activated, microglia and macrophages secreted significant levels of proMMP7. Also, conditioned medium of IL-4-activated macrophages most efficiently cleaved fibronectin aggregates upon MMP-activating conditions. Yet, coatings of MMP7-cleaved fibronectin aggregate fragments inhibited oligodendrocyte maturation, indicating that further degradation and/or clearance by phagocytosis is essential. These findings suggest that MMP7 cleaves fibronectin aggregates, while reduced (pro)MMP7 levels in MS lesions contribute to their persistent presence. Therefore, upregulating MMP7 levels may be key to remove remyelination-impairing fibronectin aggregates in MS lesions.