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.

Abstract Background Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood–brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research. However, it is unclear how iPericytes functionally compare to primary human brain vascular pericytes (HBVPs). Methods We differentiated iPSCs into iPericytes of either the mesoderm or neural crest lineage using established protocols. We compared iPericyte and HBVP morphologies, quantified gene expression by qPCR and bulk RNA sequencing, and visualised pericyte protein markers by immunocytochemistry. To determine whether the gene expression of neural crest iPericytes, mesoderm iPericytes or HBVPs correlated with their functional characteristics in vitro, we quantified EdU incorporation following exposure to the key pericyte mitogen, platelet derived growth factor (PDGF)-BB and, contraction and relaxation in response to the vasoconstrictor endothelin-1 or vasodilator adenosine, respectively. Results iPericytes were morphologically similar to HBVPs and expressed canonical pericyte markers. However, iPericytes had 1864 differentially expressed genes compared to HBVPs, while there were 797 genes differentially expressed between neural crest and mesoderm iPericytes. Consistent with the ability of HBVPs to respond to PDGF-BB signalling, PDGF-BB enhanced and a PDGF receptor-beta inhibitor impaired iPericyte proliferation. Administration of endothelin-1 led to iPericyte contraction and adenosine led to iPericyte relaxation, of a magnitude similar to the response evoked in HBVPs. We determined that neural crest iPericytes were less susceptible to PDGFR beta inhibition, but responded most robustly to vasoconstrictive mediators. Conclusions iPericytes express pericyte-associated genes and proteins and, exhibit an appropriate physiological response upon exposure to a key endogenous mitogen or vasoactive mediators. Therefore, the generation of functional iPericytes would be suitable for use in future investigations exploring pericyte function or dysfunction in neurological diseases.

Neurons have high energy demands. In a recent study, Looser et al. identified oligodendrocyte Kir4.1 as the activity-dependent driver of oligodendrocyte glycolysis that ensures that lactate is supplied to active neurons. Given that oligodendrocyte Kir4.1 also influenced axonal glucose consumption and uptake, oligodendrocytes may play a broader role in neuronal metabolic regulation.

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

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system (CNS) and the most common non-traumatic cause of neurological disability in young adults. Multiple sclerosis clinical care has improved considerably due to the development of disease-modifying therapies that effectively modulate the peripheral immune response and reduce relapse frequency. However, current treatments do not prevent neurodegeneration and disease progression, and efforts to prevent multiple sclerosis will be hampered so long as the cause of this disease remains unknown. Risk factors for multiple sclerosis development or severity include vitamin D deficiency, cigarette smoking and youth obesity, which also impact vascular health. People with multiple sclerosis frequently experience blood-brain barrier breakdown, microbleeds, reduced cerebral blood flow and diminished neurovascular reactivity, and it is possible that these vascular pathologies are tied to multiple sclerosis development. The neurovascular unit is a cellular network that controls neuroinflammation, maintains blood-brain barrier integrity, and tightly regulates cerebral blood flow, matching energy supply to neuronal demand. The neurovascular unit is composed of vessel-associated cells such as endothelial cells, pericytes and astrocytes, however neuronal and other glial cell types also comprise the neurovascular niche. Recent single-cell transcriptomics data, indicate that neurovascular cells, particular cells of the microvasculature, are compromised within multiple sclerosis lesions. Large-scale genetic and small-scale cell biology studies also suggest that neurovascular dysfunction could be a primary pathology contributing to multiple sclerosis development. Herein we revisit multiple sclerosis risk factors and multiple sclerosis pathophysiology and highlight the known and potential roles of neurovascular unit dysfunction in multiple sclerosis development and disease progression. We also evaluate the suitability of the neurovascular unit as a potential target for future disease modifying therapies for multiple sclerosis.

Alterations in upper motor neuron excitability are one of the earliest phenomena clinically detected in ALS, and in 97 % of cases, the RNA/DNA binding protein, TDP-43, is mislocalised in upper and lower motor neurons. While these are two major pathological hallmarks in disease, our understanding of where disease pathology begins, and how it spreads through the corticomotor system, is incomplete. This project used a model where mislocalised TDP-43 was expressed in the motor cortex, to determine if localised cortical pathology could result in widespread corticomotor system degeneration. Mislocalised TDP-43 caused layer V excitatory neurons in the motor cortex to become hyperexcitable after 20 days of expression. Following cortical hyperexcitability, a spread of pathogenic changes through the corticomotor system was observed. By 30 days expression, there was a significant decrease in lower motor neuron number in the lumbar spinal cord. However, cell loss occurred selectively, with a significant loss in lumbar regions 1-3, and not lumbar regions 4-6. This regional vulnerability was associated with alterations in pre-synaptic excitatory and inhibitory proteins. Excitatory inputs (VGluT2) were increased in all lumbar regions, while inhibitory inputs (GAD65/67) were increased in lumbar regions 4-6 only. This data indicates that mislocalised TDP-43 in upper motor neurons can cause lower motor neuron degeneration. Furthermore, cortical pathology increased excitatory inputs to the spinal cord, to which local circuitry compensated with an upregulation of inhibition. These findings reveal how TDP-43 mediated pathology may spread through corticofugal tracts in ALS and identify a potential pathway for therapeutic intervention.

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.

Multiple sclerosis (MS) is a complex neuroinflammatory/degenerative disease of the central nervous system (CNS) that results in the formation of demyelinated lesions and axon degeneration. MS aetiology is complex, with genetics estimated to account for ∼48% of MS risk (International Multiple Sclerosis Genetics Consortium, 2019). Despite this, families with a high incidence of MS are rare. We have generated four induced pluripotent stem cell (iPSC) lines from individuals with relapsing-remitting and secondary progressive MS within a single family. The generation of disease-specific iPSC lines from multiple members of a single family will facilitate MS genetic and functional studies.

Multiple sclerosis (MS) is a complex disease, and its pathophysiology impacts the function of immune and central nervous system cell types. Despite extensive investigation into the aetiology of MS, the underlying cause/s remain elusive and consequently, faithful in vitro or in vivo preclinical models of MS do not exist. Advances in human stem cell technologies have enabled the generation of induced pluripotent stem cells (iPSCs) from people with MS. This review summarises the discoveries made using iPSCs derived from people with MS and explores their current and potential application/s in MS research.