Euan R. Brown

Myelin sheaths comprise compacted layers of membrane wrapped around axons. Each sheath, if ‘unwrapped’, has a cytoplasm-filled space at its perimeter. Transmission electron microscopy reveals that this space contains microtubules and organelles; however, whether these are developmental remnants or serve a specific function remains unknown. Live imaging of myelinating oligodendrocytes in mice showed microtubule-dependent organelle transport within myelin sheath cytoplasmic spaces. Further, movement of myelin-located peroxisomes was modulated by neuronal electrical activity, in vitro and in vivo. Loss of oligodendroglial KIF21B and/or CNP in vivo led to the apparent stasis of myelin organelles and secondary axon pathology. We, thus, propose the acronym TRAM (transport route across myelin), defining the continuous cytoplasmic network in oligodendrocytes and myelin. This system, comprising the inner and outer tongues and paranodal loops, spans compact myelin and enables transport of metabolites and organelles between the oligodendrocyte soma and the periaxonal space.

Myelin sheaths comprise compacted layers of oligodendroglial membrane wrapped spirally around axons. Each sheath, if imagined unwrapped, has a cytoplasm-filled space at its perimeter, linking it to the oligodendrocyte soma via a short process. By electron microscopy (EM), this space, which we term the ‘myelinic channel system’ contains microtubules and membranous organelles, but whether these are remnants of development or serve a function is unknown. Performing live imaging of myelinating oligodendrocytes expressing fluorescent reporters, we found that the myelinic channel system serves microtubule-dependent organelle transport. Further, the intra-myelinic movement of peroxisomes was modulated by neuronal electrical activity in these mixed neural cell cultures. Loss of oligodendroglial Kif21b or CNP in vivo led to apparent stasis of myelin organelles and secondary axon pathology. This suggests that oligodendrocytes require motor transport in myelin to maintain axonal integrity.

Summary: Myelin sheaths comprise compacted layers of oligodendroglial membrane wrapped spirally around axons. Each sheath, if imagined unwrapped, has a cytoplasm-filled space at its perimeter, linking it to the oligodendrocyte soma via a short process. By electron microscopy (EM), this space, which we term the ‘ myelinic channel system ’ contains microtubules and membranous organelles, but whether these are remnants of development or serve a function is unknown. Performing live imaging of myelinating oligodendrocytes expressing fluorescent reporters, we found that the myelinic channel system serves microtubule-dependent organelle transport. Further, the intra-myelinic movement of peroxisomes was modulated by neuronal electrical activity in these mixed neural cell cultures. Loss of oligodendroglial Kif21b or CNP in vivo led to apparent stasis of myelin organelles and secondary axon pathology. This suggests that oligodendrocytes require motor transport in myelin to maintain axonal integrity.

The normal development and maintenance of CNS white matter, and its responses to disease and injury, are defined by synergies between axons, oligodendrocytes, astrocytes and microglia, and further influenced by peripheral components such as the gut microbiome and the endocrine and immune systems. Consequently, mechanistic insights, therapeutic approaches and safety tests rely ultimately on in vivo models and clinical trials. However, in vitro models that replicate the cellular complexity of the CNS can inform these approaches, reducing costs and minimising the use of human material or experimental animals; in line with the principles of the 3Rs. Using electrophysiology, pharmacology, time-lapse imaging, and immunological assays, we demonstrate that murine spinal cord-derived myelinating cell cultures recapitulate spinal-like electrical activity and innate CNS immune functions, including responses to disease-relevant myelin debris and pathogen associated molecular patterns (PAMPs). Further, we show they are (i) amenable to siRNA making them suitable for testing gene-silencing strategies; (ii) can be established on microelectrode arrays (MEAs) for electrophysiological studies; and (iii) are compatible with multi-well microplate formats for semi-high throughput screens, maximising information output whilst further reducing animal use. We provide protocols for each of these. Together, these advances increase the utility of this in vitro tool for studying normal and pathological development and function of white matter, and for screening therapeutic molecules or gene targets for diseases such as multiple sclerosis, motor neuron disease or spinal cord injury, whilst avoiding in vivo approaches on experimental animals.

Membrane–nanoparticle interactions are important in determining the effects of manufactured nanomaterials on cell physiology and pathology. Here, silica, titanium, zinc, and magnesium oxide nanoparticles are screened against human hERG (K_v11.1) voltage‐gated potassium channels under a whole‐cell voltage clamp. 10 µg mL⁻¹ ZnO uniquely increases the amplitude of the steady‐state current, decreases the rate of hERG current inactivation during steady‐state depolarization, accelerates channel deactivation during resurgent tail currents, and shows no significant alteration of current activation rate or voltage dependence. In contrast, ZnCl₂ causes increased current suppression with increasing concentration and fails to replicate the nanoparticle effect on decreasing inactivation. The results show a novel class of nanoparticle–biomembrane interaction involving channel gating rather than channel block, and have implications for the use of nanoparticles in biomedicine, drug delivery applications, and nanotoxicology.

Neuronal synapses are among the most scrutinized of cellular systems, serving as a model for all membrane trafficking studies. Despite this, synaptic biology has proven difficult to interrogate directly in situ due to the small size and dynamic nature of central synapses and the molecules within them. Here we determine the spatial and temporal interaction status of presynaptic proteins, imaging large cohorts of single molecules inside active synapses. Measuring rapid interaction dynamics during synaptic depolarization identified the small number of syntaxin1a and munc18-1 protein molecules required to support synaptic vesicle exocytosis. After vesicle fusion and subsequent SNARE complex disassembly, a prompt switch in syntaxin1a and munc18-1-binding mode, regulated by charge alteration on the syntaxin1a N-terminal, sequesters monomeric syntaxin1a from other disassembled fusion complex components, preventing ectopic SNARE complex formation, readying the synapse for subsequent rounds of neurotransmission.

Although mutations in the huntingtin gene (HTT) due to poly-Q expansion cause neuropathology in humans (Huntington’s disease; HD), the normal function(s) of the gene and its protein (HTT) remain obscure. With new information from recently sequenced invertebrate genomes, the study of new animal models opens the possibility of a better understanding of HTT function and its evolution. To these ends, we studied huntingtin expression pattern and dynamics in the invertebrate chordate Ciona intestinalis. Ciona huntingtin (Ci-HTT) shows a biphasic expression pattern during larval development and prior to metamorphosis. A single form of huntingtin protein is present until the early larval stages, at which time two different mass proteins become evident in the metamorphically competent larva. An antibody against Ci-HTT labeled 50 cells in the trunk mesenchyme regions in pre-hatching and hatched larvae and probably represents the distribution of the light form of the protein. Dual labeling with anti-Ci-HTT and anti-aldoketoreductase confirmed the presence of Ci-HTT in mesenchyme cells. Suppression of Ci-HTT RNA by a morpholino oligonucleotide reduced the number and apparent mobility of Ci-HTT positive cells. In Ciona, HTT expression has a dynamic temporal and spatial expression pattern that in ontogeny precedes metamorphosis. Although our results may reflect a derived function for the protein in pre- and post-metamorphic events in Ciona, we also note that as in vertebrates, there is evidence for multiple differential temporal expression, indicating that this protein probably has multiple roles in ontogeny and cell migration.