Oligodendrocyte death and myelin loss in the cuprizone model: an updated overview of the intrinsic and extrinsic causes of cuprizone demyelination.

Funder: Canadian Institutes for Health Research (CA)Funder: Natural Sciences and Engineering Research Council of Canada; doi: http://dx.doi.org/10.13039/501100000038Funder: Fondation Brain Canada; doi: http://dx.doi.org/10.13039/100009408Funder: Azrieli Foundation; doi: http://dx.doi.org/10.13039/501100005155The dietary consumption of cuprizone - a copper chelator - has long been known to induce demyelination of specific brain structures and is widely used as model of multiple sclerosis. Despite the extensive use of cuprizone, the mechanism by which it induces demyelination are still unknown. With this review we provide an updated understanding of this model, by showcasing two distinct yet overlapping modes of action for cuprizone-induced demyelination; 1) damage originating from within the oligodendrocyte, caused by mitochondrial dysfunction or reduced myelin protein synthesis. We term this mode of action 'intrinsic cell damage'. And 2) damage to the oligodendrocyte exerted by inflammatory molecules, brain resident cells, such as oligodendrocytes, astrocytes, and microglia or peripheral immune cells - neutrophils or T-cells. We term this mode of action 'extrinsic cellular damage'. Lastly, we summarize recent developments in research on different forms of cell death induced by cuprizone, which could add valuable insights into the mechanisms of cuprizone toxicity. With this review we hope to provide a modern understanding of cuprizone-induced demyelination to understand the causes behind the demyelination in MS

Apoptosis of Oligodendrocytes during Early Development Delays Myelination and Impairs Subsequent Responses to Demyelination.

During mammalian development, myelin-forming oligodendrocytes are generated and axons ensheathed according to a tightly regulated sequence of events. Excess premyelinating oligodendrocytes are eliminated by apoptosis and the timing of the onset of myelination in any specific CNS region is highly reproducible. Although the developing CNS recovers more effectively than the adult CNS from similar insults, it is unknown whether early loss of oligodendrocyte lineage cells leads to long-term functional deficits. To directly assess whether the loss of oligodendrocytes during early postnatal spinal cord development impacted oligodendrogenesis, myelination, and remyelination, transgenic mouse lines were generated in which a modified caspase-9 molecule allowed spatial and temporal control of the apoptotic pathway specifically in mature, myelin basic protein expressing oligodendrocytes (MBP-iCP9). Activating apoptosis in MBP(+) cells of the developing spinal cord during the first postnatal week inhibited myelination. This inhibition was transient, and the levels of myelination largely returned to normal after 2 weeks. Despite robust developmental plasticity, MBP-iCP9-induced oligodendrocyte apoptosis compromised the rate and extent of adult remyelination. Remyelination failure correlated with a truncated proliferative response of oligodendrocyte progenitor cells, suggesting that depleting the oligodendrocyte pool during critical developmental periods compromises the regenerative response to subsequent demyelinating lesions. SIGNIFICANCE STATEMENT This manuscript demonstrates that early insults leading to oligodendrocyte apoptosis result in the impairment of recovery from demyelinating diseases in the adult. These studies begin to provide an initial understanding of the potential failure of recovery in insults, such as periventricular leukomalacia and multiple sclerosis

Nile Red fluorescence spectroscopy reports early physicochemical changes in myelin with high sensitivity

The molecular composition of myelin membranes determines their structure and function. Even minute changes to the biochemical balance can have profound consequences for axonal conduction and the synchronicity of neural networks. Hypothesizing that the earliest indication of myelin injury involves changes in the composition and/or polarity of its constituent lipids, we developed a sensitive spectroscopic technique for defining the chemical polarity of myelin lipids in fixed frozen tissue sections from rodent and human. The method uses a simple staining procedure involving the lipophilic dye Nile Red, whose fluorescence spectrum varies according to the chemical polarity of the microenvironment into which the dye embeds. Nile Red spectroscopy identified histologically intact yet biochemically altered myelin in prelesioned tissues, including mouse white matter following subdemyelinating cuprizone intoxication, as well as normal-appearing white matter in multiple sclerosis brain. Nile Red spectroscopy offers a relatively simple yet highly sensitive technique for detecting subtle myelin changes

Unique spectral signatures of the nucleic acid dye acridine orange can distinguish cell death by apoptosis and necroptosis

Cellular injury and death are ubiquitous features of disease, yet tools to detect them are limited and insensitive to subtle pathological changes. Acridine orange (AO), a nucleic acid dye with unique spectral properties, enables real-time measurement of RNA and DNA as proxies for cell viability during exposure to various noxious stimuli. This tool illuminates spectral signatures unique to various modes of cell death, such as cells undergoing apoptosis versus necrosis/ necroptosis. This new approach also shows that cellular RNA decreases during necrotic, necroptotic, and apoptotic cell death caused by demyelinating, ischemic, and traumatic injuries, implying its involvement in a wide spectrum of tissue pathologies. Furthermore, cells with pathologically low levels of cytoplasmic RNA are detected earlier and in higher numbers than with standard markers including TdT-mediated dUTP biotin nick-end labeling and cleaved caspase 3 immunofluorescence. Our technique highlights AO-labeled cytoplasmic RNA as an important early marker of cellular injury and a sensitive indicator of various modes of cell death in a range of experimental models

Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy

For decades lysophosphatidylcholine (LPC, lysolecithin) has been used to induce demyelination, without a clear understanding of its mechanisms. LPC is an endogenous lysophospholipid so it may cause demyelination in certain diseases. We investigated whether known receptor systems, inflammation or nonspecific lipid disruption mediates LPC-demyelination in mice. We found that LPC nonspecifically disrupted myelin lipids. LPC integrated into cellular membranes and rapidly induced cell membrane permeability; in mice, LPC injury was phenocopied by other lipid disrupting agents. Interestingly, following its injection into white matter, LPC was cleared within 24 hr but by five days there was an elevation of endogenous LPC that was not associated with damage. This elevation of LPC in the absence of injury raises the possibility that the brain has mechanisms to buffer LPC. In support, LPC injury in culture was significantly ameliorated by albumin buffering. These results shed light on the mechanisms of LPC injury and homeostasis

CNS Myelin Wrapping Is Driven by Actin Disassembly

Myelin is essential in vertebrates for the rapid propagation of action potentials, but the molecular mechanisms driving its formation remain largely unknown. Here we show that the initial stage of process extension and axon ensheathment by oligodendrocytes requires dynamic actin filament assembly by the Arp2/3 complex. Unexpectedly, subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins and rapid disassembly of the oligodendrocyte actin cytoskeleton and does not require Arp2/3. Inducing loss of actin filaments drives oligodendrocyte membrane spreading and myelin wrapping in vivo, and the actin disassembly factor gelsolin is required for normal wrapping. We show that myelin basic protein, a protein essential for CNS myelin wrapping whose role has been unclear, is required for actin disassembly, and its loss phenocopies loss of actin disassembly proteins. Together, these findings provide insight into the molecular mechanism of myelin wrapping and identify it as an actin-independent form of mammalian cell motility

Single-cell microglial transcriptomics during demyelination defines a microglial state required for lytic carcass clearance

Abstract Background Microglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood. Methods To understand how microglia respond during demyelination, we fed mice cuprizone—a potent demyelinating agent—and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response. Results The cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination. Conclusions Microglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state