長内 康幸

Myelin formation by oligodendrocytes is essential for the regulation of the conduction velocity and proper brain function. To ensure accurate information processing in response to experiences such as sensory stimuli and learning, oligodendrocytes adjust their number and morphology. In addition, oligodendrocyte morphology changes with senescence and in the presence of neurodegenerative diseases. Thus, visualizing oligodendrocytes and analyzing their morphology is crucial for understanding how our brains change under such conditions. Herein, we describe the methods for labeling and analyzing the morphologies of individual oligodendrocytes in mouse white matter at the light microscopic level. PDGFRa-CreERT2:Tau-mGFP and PLP-CreERT2:Tau-mGFP mice enable us to visualize and analyze later-born or early-born oligodendrocyte morphology. In addition, sparse oligodendrocyte labeling with attenuated rabies virus expressing GFP enables the visualization and morphological analysis of individual oligodendrocytes in various brain white matter regions without the need for transgenic animals. Furthermore, the combination with immunostaining in thick tissues enables the identification of labeled oligodendrocytes and myelin sheaths, as well as their interactions with neuronal axons. These methods are suitable for revealing how oligodendrocytes adapt their morphologies depending on environmental stimuli or pathological conditions.

The activity of oligodendrocyte progenitor cells (OPCs) and oligodendrocytes (OLs) throughout life drives myelination, which is crucial for rapid neuronal communication. OLs in the aging brain demonstrate a reduced capacity for myelin formation and maintenance, but the underlying differentiation of individual OLs and morphological changes of their myelin in aging remain unclear. Here, we utilized Pdgfra-CreERT2:Tau-mGFP double transgenic mice to selectively label and visualize newly generated OLs in aged (78-week-old) mice and compared them with those in young (8-week-old) mice. We revealed a significantly lower percentage of newly generated OLs that differentiated into mature OLs and a decreased rate of myelinating OLs accumulation in aged mice compared with young mice. Additionally, newly generated myelinating mature OLs in aged mice demonstrated significantly greater height compared with those in young mice. Furthermore, myelin internodes were significantly shorter and significantly fewer in aged mice compared with young mice. Our results indicate age-related impairments in the differentiation efficiency of aged OPCs and age-related morphological changes in OLs. These alterations in newly generated OLs may contribute to impaired myelination, reduced myelin turnover, and disrupted myelin maintenance in aged mice.

White matter injury is caused by cerebral blood flow disturbances associated with stroke and demyelinating diseases such as multiple sclerosis. Remyelination is induced spontaneously after white matter injury, but progressive multiple sclerosis and white matter stroke are usually characterised by remyelination failure. However, the mechanisms underlying impaired remyelination in lesions caused by demyelination and stroke remain unclear. In the current study, we demonstrated that collagen fibres accumulated in the demyelinated lesions of multiple sclerosis patients (age range 23-80 years) and white matter lesions of stroke patients (age range 80-87 years), suggesting that the accumulation of collagen fibres correlates with remyelination failure in these lesions. To investigate the function of collagen fibres in the white matter lesions, we generated two types of white matter injury in mice. We induced focal demyelination by lysolecithin (LPC) injection and ischemic stroke by endothelin 1 (ET1) injection into the internal capsule. We found that type I collagen fibres were secreted in ET1-induced lesions with impaired white matter regeneration in the chronic phase of disease. We also showed that monocyte-derived macrophages that infiltrated into lesions from the peripheral blood produced type I collagen after white matter injury, and that type I collagen also exacerbated microglial activation, astrogliosis, and axonal injury. Finally, we demonstrated that oligodendrocyte differentiation and remyelination were inhibited in the presence of type I collagen after LPC-induced demyelination. These results suggest that type I collagen secreted by monocyte-derived macrophages inhibited white matter regeneration, and therefore, the modulation of type I collagen metabolism might be a novel therapeutic target for white matter injury.

Myelin formation by oligodendrocytes regulates the conduction velocity and functional integrity of neuronal axons. While individual oligodendrocytes form myelin sheaths around multiple axons and control the functions of neural circuits where the axons are involved, it remains unclear if oligodendrocytes selectively form myelin sheaths around specific subtypes of axons. Using the combination of rabies virus-mediated single oligodendrocyte labeling and immunostaining with tissue clearing, we revealed that approximately half of the oligodendrocytes preferentially myelinate axons originating from Purkinje cells in the white matter of adult mouse cerebella. The preference for Purkinje cell axons was more pronounced during development when the process of myelination within cerebellar white matter was initiated; over 90% of oligodendrocytes preferentially myelinated Purkinje cell axons. Preferential myelination of Purkinje cell axons was further confirmed by immuno-electron microscopy and transgenic mice that label early-born oligodendrocytes. Transgenic mice that label oligodendrocytes differentiated at the early development showed that early-born oligodendrocytes preferentially myelinate Purkinje cell axons in the matured cerebellar white matter. In contrast, transgenic mice that label oligodendrocytes differentiated after the peak of cerebellar myelination showed that the later-differentiated oligodendrocytes dominantly myelinated non-Purkinje cell axons. These results demonstrate that a significant proportion of oligodendrocytes preferentially myelinate functionally distinct axons in the cerebellar white matter, and the axonal preference of myelination by individual oligodendrocytes is established depending on the timing of their differentiation during development. Our data provide the evidence that there is a critical time window of myelination that a specific subtype of axons are dominantly myelinated by the oligodendrocytes.