Choroid plexuses carry nodal-like cilia that undergo axoneme regression from early adult stage.

Choroid plexuses (ChPs) produce cerebrospinal fluid and sense non-cell-autonomous stimuli to control the homeostasis of the central nervous system. They are mainly composed of epithelial multiciliated cells, whose development and function are still controversial. We have thus characterized the stepwise order of mammalian ChP epithelia cilia formation using a combination of super-resolution-microscopy approaches and mouse genetics. We show that ChP ciliated cells are built embryonically on a treadmill of spatiotemporally regulated events, starting with atypical centriole amplification and ending with the construction of nodal-like 9+0 cilia, characterized by both primary and motile features. ChP cilia undergo axoneme resorption at early postnatal stages through a microtubule destabilization process controlled by the microtubule-severing enzyme spastin and mitigated by polyglutamylation levels. Notably, this phenotype is preserved in humans, suggesting a conserved ciliary resorption mechanism in mammals.

The Role of Lamins in the Nucleoplasmic Reticulum, a Pleiomorphic Organelle That Enhances Nucleo-Cytoplasmic Interplay.

Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer's disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.

Progressive axonopathy when oligodendrocytes lack the myelin protein CMTM5.

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here, we find that expression of the tetraspan-transmembrane protein CMTM5 (chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and central nervous system (CNS) myelin. Genetic disruption of the Cmtm5 gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5 deficiency causes an early-onset progressive axonopathy, which we also observe in global and tamoxifen-induced oligodendroglial Cmtm5 mutants. Presence of the WldS mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.

White matter integrity in mice requires continuous myelin synthesis at the inner tongue.

Myelin, the electrically insulating sheath on axons, undergoes dynamic changes over time. However, it is composed of proteins with long lifetimes. This raises the question how such a stable structure is renewed. Here, we study the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survive recombination, continue to express myelin genes, but they fail to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualize myelin-like membranes failing to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicates degradation of existing myelin at the abaxonal side and the inner tongue of the sheath. Thinning of compact myelin and shortening of internodes result in the loss of about 50% of myelin and axonal pathology within 20 weeks post recombination. In summary, our data suggest that functional axon-myelin units require the continuous incorporation of new myelin membranes.

Microglia facilitate repair of demyelinated lesions via post-squalene sterol synthesis.

The repair of inflamed, demyelinated lesions as in multiple sclerosis (MS) necessitates the clearance of cholesterol-rich myelin debris by microglia/macrophages and the switch from a pro-inflammatory to an anti-inflammatory lesion environment. Subsequently, oligodendrocytes increase cholesterol levels as a prerequisite for synthesizing new myelin membranes. We hypothesized that lesion resolution is regulated by the fate of cholesterol from damaged myelin and oligodendroglial sterol synthesis. By integrating gene expression profiling, genetics and comprehensive phenotyping, we found that, paradoxically, sterol synthesis in myelin-phagocytosing microglia/macrophages determines the repair of acutely demyelinated lesions. Rather than producing cholesterol, microglia/macrophages synthesized desmosterol, the immediate cholesterol precursor. Desmosterol activated liver X receptor (LXR) signaling to resolve inflammation, creating a permissive environment for oligodendrocyte differentiation. Moreover, LXR target gene products facilitated the efflux of lipid and cholesterol from lipid-laden microglia/macrophages to support remyelination by oligodendrocytes. Consequently, pharmacological stimulation of sterol synthesis boosted the repair of demyelinated lesions, suggesting novel therapeutic strategies for myelin repair in MS.

A role of oligodendrocytes in information processing.

Myelinating oligodendrocytes enable fast propagation of action potentials along the ensheathed axons. In addition, oligodendrocytes play diverse non-canonical roles including axonal metabolic support and activity-dependent myelination. An open question remains whether myelination also contributes to information processing in addition to speeding up conduction velocity. Here, we analyze the role of myelin in auditory information processing using paradigms that are also good predictors of speech understanding in humans. We compare mice with different degrees of dysmyelination using acute multiunit recordings in the auditory cortex, in combination with behavioral readouts. We find complex alterations of neuronal responses that reflect fatigue and temporal acuity deficits. We observe partially discriminable but similar deficits in well myelinated mice in which glial cells cannot fully support axons metabolically. We suggest a model in which myelination contributes to sustained stimulus perception in temporally complex paradigms, with a role of metabolically active oligodendrocytes in cortical information processing.

Transmission Electron Microscopy of Oligodendrocytes and Myelin.

In this chapter, we describe protocols to study different aspects of oligodendrocytes and myelin using electron microscopy. First, we describe in detail how to prepare central nervous system tissue routinely by perfusion fixation of the animal and conventional embedding in Epon resin. Then, we explain how, with some modifications, chemically fixed tissue can be used for immunoelectron microscopy on cryosections. Chemical fixation and Epon embedding can also be applied to purified myelin to assess the quality of the preparation. Furthermore, we describe how cryopreparation by high-pressure freezing can be used to study the fine structure of myelin in nerve, brain, and spinal cord tissue. The differences in the structural appearance of oligodendrocytes and myelin between cryopreserved and conventionally processed samples are compared using representative images. Since primary cultured oligodendrocytes are used to study structure and function in vitro, we provide protocols for chemical fixation and Epon embedding of these cultures. Finally, we explain how the cytoskeleton of cultured oligodendrocytes can be visualized by using transmission electron microscopy on platinum-carbon replicas. In this chapter, we provide a wide range of protocols that can be applied to shed light on the different biological aspects of myelin and oligodendrocytes.

Y-P30 promotes axonal growth by stabilizing growth cones.

The 30-amino acid peptide Y-P30, generated from the N-terminus of the human dermcidin precursor protein, has been found to promote neuronal survival, cell migration and neurite outgrowth by enhancing the interaction of pleiotrophin and syndecan-3. We now show that Y-P30 activates Src kinase and extracellular signal-regulated kinase (ERK). Y-P30 promotes axonal growth of mouse embryonic stem cell-derived neurons, embryonic mouse spinal cord motoneurons, perinatal rat retinal neurons, and rat cortical neurons. Y-P30-mediated axon growth was dependent on heparan sulfate chains. Y-P30 decreased the proportion of collapsing/degenerating growth cones of cortical axons in an Src and ERK-dependent manner. Y-P30 increased for 90 min in axonal growth cones the level of Tyr418-phosphorylated Src kinase and the amount of F-actin, and transiently the level of Tyr-phosphorylated ERK. Levels of total Src kinase, actin, GAP-43, cortactin and the glutamate receptor subunit GluN2B were not altered. When exposed to semaphorin-3a, Y-P30 protected a significant fraction of growth cones of cortical neurons from collapse. These results suggest that Y-P30 promotes axonal growth via Src- and ERK-dependent mechanisms which stabilize growth cones and confer resistance to collapsing factors.