Oxysterols, oxidized derivatives of cholesterol, are key modulators of central nervous system (CNS) function. Acting as both signaling molecules and cytotoxic mediators, oxysterols can influence glial activity, neuroinflammation and myelination. Glial cells (astrocytes, oligodendrocytes, and microglia) maintain neuronal homeostasis, support synaptic plasticity and mediate CNS repair. Dysregulated glial function contributes to chronic neuroinflammation, a hallmark of neurodegenerative and neuropsychiatric disorders such as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. In this review article, we discuss the roles of oxysterols in the CNS with a particular focus on their impact on glial cells, neuroinflammation, myelination and neural circuit function. Therapeutically targeting oxysterol metabolism may mitigate oxidative stress, limit apoptosis, enhance glial health and influence myelin repair. Elucidating oxysterol signaling in glia and neurons provides critical insight into neural circuit regulation and identifies promising strategies for treating demyelinating, neurodegenerative and neuropsychiatric disorders.

Myelination of the developing central nervous system (CNS) increases action potential conduction velocity but can also act as a plasticity brake, limiting neurite reorganisation and synaptic plasticity. As myelination is a life-long process, the myelin laid down in development or adulthood could also affect synapse number or plasticity across the lifespan. We report that conditionally deleting the transcription factor, myelin regulatory factor (Myrf), to disrupt the myelin maintenance program in oligodendrocytes (OLs) from P57 (Plp-CreERT2 :: Myrf fl/fl mice), led to significant myelin loss and impaired action potential conduction and gross motor performance. By sparsely labelling layer V pyramidal neurons in the primary motor cortex, we could visualise the apical and basal dendrites and their excitatory post-synaptic dendritic spines and determined that spine density was normal in P57+60 Plp-CreERT2 :: Myrf fl/fl mice. However, a higher proportion of the dendritic spines were large, stable mushroom spines and generated larger amplitude miniature excitatory post-synaptic currents. These data indicate that the myelin maintenance program is critical for preserving neuron adaptability, homeostasis and glutamate sensitivity. When we instead prevented the addition of new OLs and myelin from P57, action potential conduction velocity and gross motor performance appeared normal. Spine density was also normal in Pdgfrα-CreERTM :: Myrf fl/fl mice, however, spine morphology was changed along the basal dendrites of layer V M1 pyramidal neurons. At P117, the basal dendrites were frozen in a state that resembled P57 dendrites, as they retained a higher proportion of thin, plastic spines. These data suggest that adult myelination supports the life-long accumulation of stable spines within the basal but not apical dendritic compartment and have important implications for understanding dendritic plasticity rules in the motor circuit. ### Competing Interest Statement The authors have declared no competing interest.

Capillary pericytes are important regulators of cerebral blood flow, blood-brain barrier integrity and neuroinflammation, but can become lost or dysfunctional in disease. The consequences of pericyte loss or dysfunction is extremely difficult to discern when it forms one component of a complex disease process. To evaluate this directly, we examined the effect of adult pericyte loss on mouse voluntary movement and motor function, and physiological responses such as hypoxia, blood-brain barrier (BBB) integrity and glial reactivity. Tamoxifen delivery to Pdgfr beta-CreER(T2):: Rosa26-DTA transgenic mice was titrated to produce a dose-dependent ablation of pericytes in vivo. 100mg/kg of tamoxifen ablated approximately half of all brain pericytes, while two consecutive daily doses of 300mg/kg tamoxifen ablated >80% of brain pericytes. In the open field test, mice with similar to 50% pericyte loss spent more time immobile and travelled half the distance of control mice. Mice with >80% pericyte ablation also slipped more frequently while performing the beam walk task. Our histopathological analyses of the brain revealed that blood vessel density was unchanged, but vessel lumen width was increased. Pericyte-ablated mice also exhibited: mild BBB disruption; increased neuronal hypoxia; astrogliosis and increased IBA1(+) immunoreactivity, suggestive of microgliosis and/or macrophage infiltration. Our results highlight the importance of pericytes in the brain, as pericyte loss can directly compromise brain health and induce behavioural alterations in mice.

Oligodendrocyte progenitor cells (OPCs) receive synaptic input from a diverse range of neurons in the developing and adult brain. Understanding whether the neuronal populations that synapse with OPCs in the healthy brain is altered by demyelination and/or remyelination may support the advancement of neuroprotective or myelin repair strategies being developed for demyelinating diseases such as multiple sclerosis. To explore this possibility, we employed cre-lox transgenic technology to facilitate the infection of OPCs by a modified rabies virus, enabling the retrograde monosynaptic tracing of neuron→OPC connectivity. In the healthy adult mouse, OPCs in the corpus callosum primarily received synaptic input from ipsilateral cortical neurons. Of the cortical neurons, ∼50% were layer V pyramidal cells. Cuprizone demyelination reduced the total number of labeled neurons. However, the frequency/kinetics of mini-excitatory postsynaptic currents recorded from OPCs appeared preserved. Of particular interest, demyelination increased the number of labeled layer II/III pyramidal neurons and also increased at the expense of layer V pyramidal neurons, a change that was largely ameliorated by remyelination. These data suggest that in the healthy adult mouse brain, callosal OPCs primarily receive synaptic input from cortical layer V pyramidal neurons. However, callosal demyelination is associated with a population switch and OPCs equally synapse with layer II/III and V pyramidal neurons to synapse with OPCs, until myelin is restored.

In people with multiple sclerosis (MS), newborn and surviving oligodendrocytes (OLs) can contribute to remyelination, however, current therapies are unable to enhance or sustain endogenous repair. Low intensity repetitive transcranial magnetic stimulation (LI-rTMS), delivered as an intermittent theta burst stimulation (iTBS), increases the survival and maturation of newborn OLs in the healthy adult mouse cortex, but it is unclear whether LI-rTMS can promote remyelination. To examine this possibility, we fluorescently labelled oligodendrocyte progenitor cells (OPCs; Pdgfrα-CreER transgenic mice) or mature OLs (Plp-CreER transgenic mice) in the adult mouse brain and traced the fate of each cell population over time. Daily sessions of iTBS (600 pulses; 120 mT), delivered during cuprizone (CPZ) feeding, did not alter new or pre-existing OL survival but increased the number of myelin internodes elaborated by new OLs in the primary motor cortex (M1). This resulted in each new M1 OL producing 471 µm more myelin. When LI-rTMS was delivered after CPZ withdrawal (during remyelination), it significantly increased the length of the internodes elaborated by new M1 and callosal OLs, increased the number of surviving OLs that supported internodes in the corpus callosum (CC), and increased the proportion of axons that were myelinated. The ability of LI-rTMS to modify cortical neuronal activity and the behaviour of new and surviving OLs, suggests that it may be a suitable adjunct intervention to enhance remyelination in people with MS.

Glutamate-gated kainate receptors comprising the Gluk4 subunit (encoded by Grik4 ) are highly expressed by neurons in the central nervous system. We report that Grik4 mRNA is widely expressed by neurons in the adult mouse motor cortex, where GluK4-containing kainate receptors account for ∼60% of the kainate evoked current in layer V pyramidal neurons. To elucidate their role in motor circuit regulation, we analysed the behaviour of mice that lacked the pore forming domain of the GluK4 subunit ( Grik4-/- mice). Grik4-/- mice were hyperactive, had an abnormal gait, and impaired motor coordination. At postnatal day (P)60, layer V pyramidal neurons received fewer miniature excitatory post synaptic currents, had a reduced density of thin spines on their basal dendrites, and a reduced density of VGlut1 puncta at the soma, consistent with neurons receiving fewer excitatory synaptic connections. Grik4-/- mice also lost ∼44% of their callosal axons between P60 and P180 and the amplitude of the callosal compound action potential was reduced by ∼25-30%. RNA sequencing data support the capacity for Grik4 to modulate synaptic and neuroprotective signalling pathways.### Competing Interest StatementThe authors have declared no competing interest.

Background Low-intensity repetitive transcranial magnetic stimulation (rTMS), delivered as a daily intermittent theta burst stimulation (iTBS) for four consecutive weeks, increased the number of new oligodendrocytes in the adult mouse brain. Therefore, rTMS holds potential as a remyelinating intervention for people with multiple sclerosis (MS). Objective Primarily to determine the safety and tolerability of our rTMS protocol in people with MS. Secondary objectives include feasibility, blinding and an exploration of changes in magnetic resonance imaging (MRI) metrics, patient-reported outcome measures (PROMs) and cognitive or motor performance. Methods A randomised (2:1), placebo controlled, single blind, parallel group, phase 1 trial of 20 rTMS sessions (600 iTBS pulses per hemisphere; 25% maximum stimulator output), delivered over 4–5 weeks. Twenty participants were randomly assigned to ‘sham’ ( n = 7) or active rTMS ( n = 13), with the coil positioned at 90° or 0°, respectively. Results Five adverse events (AEs) including one serious AE reported. None were related to treatment. Protocol compliance was high (85%) and blinding successful. Within participant MRI metrics, PROMs and cognitive or motor performance were unchanged over time. Conclusion Twenty sessions of rTMS is safe and well tolerated in a small group of people with MS. The study protocol and procedures are feasible. Improvement of sham is warranted before further investigating safety and efficacy.

Multiple sclerosis (MS) is a chronic neurological condition and the leading cause of non-traumatic disability in young adults. MS pathogenesis leads to the death of oligodendrocytes, demyelination, and progressive central nervous system neurodegeneration. Endogenous remyelination occurs in people with MS (PwMS) but is insufficient to repair the damage. Our preclinical studies in mice indicate that endogenous remyelination can be supported by the delivery of repetitive transcranial magnetic stimulation (rTMS). Our phase I trial concluded that 20 sessions of rTMS, delivered over 5 weeks, are safe and feasible for PwMS. This phase II trial aims to investigate the safety and preliminary efficacy of rTMS for PwMS. Participants must be aged 18–65 years, diagnosed with MS by a neurologist, stable and relapse free for 6 months, have an Extended Disability Status Scale (EDSS) between 1.5 and 6 (inclusive), willing to travel to a study site every weekday for 4 consecutive weeks, and able to provide informed consent and access the internet. Participants from multiple centres will be randomised 2:1 (rTMS to sham) stratified by sex. The intervention will be delivered with a Magstim Rapid2 stimulator device and circular 90-mm coil or MagVenture MagPro stimulator device with C100 circular coil, positioned to stimulate a broad area including frontal and parietal cortices. For the rTMS group, pulse intensity will be set at 18

Abstract Background Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease, characterised by oligodendrocyte death and demyelination. Oligodendrocyte progenitor cells can differentiate into new replacement oligodendrocytes; however, remyelination is insufficient to protect neurons from degeneration in people with MS. We previously reported that 4 weeks of daily low-intensity repetitive transcranial magnetic stimulation (rTMS) in an intermittent theta-burst stimulation (iTBS) pattern increased the number of new myelinating oligodendrocytes in healthy adult mice. This study translates this rTMS protocol and aims to determine its safety and tolerability for people living with MS. We will also perform magnetic resonance imaging (MRI) and symptom assessments as preliminary indicators of myelin addition following rTMS. Methods Participants (N = 30, aged 18–65 years) will have a diagnosis of relapsing-remitting or secondary progressive MS. ≤2 weeks before the intervention, eligible, consenting participants will complete a physical exam, baseline brain MRI scan and participant-reported MS symptom assessments [questionnaires: Fatigue Severity Scale, Quality of Life (AQoL-8D), Hospital Anxiety and Depression Scale; and smartphone-based measures of cognition (electronic symbol digit modalities test), manual dexterity (pinching test, draw a shape test) and gait (U-Turn test)]. Participants will be pseudo-randomly allocated to rTMS (n=20) or sham (placebo; n=10), stratified by sex. rTMS or sham will be delivered 5 days per week for 4 consecutive weeks (20 sessions, 6 min per day). rTMS will be applied using a 90-mm circular coil at low-intensity (25% maximum stimulator output) in an iTBS pattern. For sham, the coil will be oriented 90° to the scalp, preventing the magnetic field from stimulating the brain. Adverse events will be recorded daily. We will evaluate participant blinding after the first, 10th and final session. After the final session, participants will repeat symptom assessments and brain MRI, for comparison with baseline. Participant-reported assessments will be repeated at 4-month post-allocation follow-up. Discussion This study will determine whether this rTMS protocol is safe and tolerable for people with MS. MRI and participant-reported symptom assessments will serve as preliminary indications of rTMS efficacy for myelin addition to inform further studies. Trial registration Australian New Zealand Clinical Trials Registry ACTRN12619001196134. Registered on 27 August 2019

Oligodendrocyte progenitor cells (OPCs) express protocadherin 15 (Pcdh15), a member of the cadherin superfamily of transmembrane proteins. Little is known about the function of Pcdh15 in the central nervous system (CNS), however, Pcdh15 expression can predict glioma aggression and promote the separation of embryonic human OPCs immediately following a cell division. Herein, we show that Pcdh15 knockdown significantly increases extracellular signal-related kinase (ERK) phosphorylation and activation to enhance OPC proliferation in vitro. Furthermore, Pcdh15 knockdown elevates Cdc42-Arp2/3 signalling and impairs actin kinetics, reducing the frequency of lamellipodial extrusion and slowing filopodial withdrawal. Pcdh15 knockdown also reduces the number of processes supported by each OPC and new process generation. Our data indicate that Pcdh15 is a critical regulator of OPC proliferation and process motility, behaviours that characterise the function of these cells in the healthy CNS, and provide mechanistic insight into the role that Pcdh15 might play in glioma progression.

Central nervous system myelination increases action potential conduction velocity. However, it is unclear how myelination is coordinated to ensure the temporally precise arrival of action potentials and facilitate information processing within cortical and associative circuits. Here, we show that myelin sheaths, supported by mature oligodendrocytes, remain plastic in the adult mouse brain and undergo subtle structural modifications to influence action potential conduction velocity. Repetitive transcranial magnetic stimulation and spatial learning, two stimuli that modify neuronal activity, alter the length of the nodes of Ranvier and the size of the periaxonal space within active brain regions. This change in the axon-glial configuration is independent of oligodendrogenesis and robustly alters action potential conduction velocity. Because aptitude in the spatial learning task was found to correlate with action potential conduction velocity in the fimbria-fornix pathway, modifying the axon-glial configuration may be a mechanism that facilitates learning in the adult mouse brain.

Myelin and axon losses are associated with cognitive decline in healthy ageing but are worse in people diagnosed with tauopathy. To determine whether tauopathy is also associated with enhanced myelin plasticity, we evaluated the behaviour of OPCs in mice that expressed a human pathological variant of microtubule-associated protein tau (MAPT(P301S)). By 6 months of age (P180), MAPT(P301S) mice overexpressed hyperphosphorylated tau and had developed reactive gliosis in the hippocampus but had not developed overt locomotor or memory impairment. By performing cre-lox lineage tracing of adult OPCs, we determined that the number of newborn oligodendrocytes added to the hippocampus, entorhinal cortex and fimbria was equivalent in control and MAPT(P301S) mice prior to P150. However, between P150 and P180, significantly more new oligodendrocytes were added to these regions in the MAPT(P301S) mouse brain. This large increase in new oligodendrocyte number was not the result of increased OPC proliferation, nor did it alter oligodendrocyte density in the hippocampus, entorhinal cortex or fimbria, which was equivalent in P180 wild-type and MAPT(P301S) mice. Furthermore, the proportion of hippocampal and fimbria axons with myelin was unaffected by tauopathy. However, the proportion of myelinated axons that were ensheathed by immature myelin internodes was significantly increased in the hippocampus and fimbria of P180 MAPT(P301S) mice, when compared with their wild-type littermates. These data suggest that MAPT(P301S) transgenic mice experience significant oligodendrocyte turnover, with newborn oligodendrocytes compensating for myelin loss early in the development of tauopathy.

During cortical development and throughout adulthood, oligodendrocytes add myelin internodes to glutamatergic projection neurons and GABAergic inhibitory neurons. In addition to directing node of Ranvier formation, to enable saltatory conduction and influence action potential transit time, oligodendrocytes support axon health by communicating with axons via the periaxonal space and providing metabolic support that is particularly critical for healthy ageing. In this review we outline the timing of oligodendrogenesis in the developing mouse and human cortex and describe the important role that oligodendrocytes play in sustaining and modulating neuronal function. We also provide insight into the known and speculative impact that myelination has on cortical axons and their associated circuits during the developmental critical periods and throughout life, particularly highlighting their life-long role in learning and remembering.