Repression of developmental transcription factor networks triggers aging-associated gene expression in human glial progenitor cells.

Human glial progenitor cells (hGPCs) exhibit diminished expansion competence with age, as well as after recurrent demyelination. Using RNA-sequencing to compare the gene expression of fetal and adult hGPCs, we identify age-related changes in transcription consistent with the repression of genes enabling mitotic expansion, concurrent with the onset of aging-associated transcriptional programs. Adult hGPCs develop a repressive transcription factor network centered on MYC, and regulated by ZNF274, MAX, IKZF3, and E2F6. Individual over-expression of these factors in iPSC-derived hGPCs lead to a loss of proliferative gene expression and an induction of mitotic senescence, replicating the transcriptional changes incurred during glial aging. miRNA profiling identifies the appearance of an adult-selective miRNA signature, imposing further constraints on the expansion competence of aged GPCs. hGPC aging is thus associated with acquisition of a MYC-repressive environment, suggesting that suppression of these repressors of glial expansion may permit the rejuvenation of aged hGPCs.

Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain.

Neonatally transplanted human glial progenitor cells (hGPCs) can myelinate the brains of myelin-deficient shiverer mice, rescuing their phenotype and survival. Yet, it has been unclear whether implanted hGPCs are similarly able to remyelinate the diffusely demyelinated adult CNS. We, therefore, ask if hGPCs could remyelinate both congenitally hypomyelinated adult shiverers and normal adult mice after cuprizone demyelination. In adult shiverers, hGPCs broadly disperse and differentiate as myelinating oligodendrocytes after subcortical injection, improving both host callosal conduction and ambulation. Implanted hGPCs similarly remyelinate denuded axons after cuprizone demyelination, whether delivered before or after demyelination. RNA sequencing (RNA-seq) of hGPCs back from cuprizone-demyelinated brains reveals their transcriptional activation of oligodendrocyte differentiation programs, while distinguishing them from hGPCs not previously exposed to demyelination. These data indicate the ability of transplanted hGPCs to disperse throughout the adult CNS, to broadly myelinate regions of dysmyelination, and also to be recruited as myelinogenic oligodendrocytes later in life, upon demyelination-associated demand.

Remyelinating Oligodendrocyte Precursor Cell miRNAs from the Sfmbt2 Cluster Promote Cell Cycle Arrest and Differentiation.

Oligodendrocyte (OL) loss contributes to the functional deficits underlying diseases with a demyelinating component. Remyelination by oligodendrocyte progenitor cells (OPCs) can restore these deficits. To understand the role that microRNAs (miRNAs) play in remyelination, 2',3'-cyclic-nucleotide 3'-phosphodiesterase-EGFP(+) mice were treated with cuprizone, and OPCs were sorted from the corpus callosum. Microarray analysis revealed that Sfmbt2 family miRNAs decreased during cuprizone treatment. One particular Sfmbt2 miRNA, miR-297c-5p, increased during mouse OPC differentiation in vitro and during callosal development in vivo. When overexpressed in both mouse embryonic fibroblasts and rat OPCs (rOPCs), cell cycle analysis revealed that miR-297c-5p promoted G1/G0 arrest. Additionally, miR-297c-5p transduction increased the number of O1(+) rOPCs during differentiation. Luciferase reporter assays confirmed that miR-297c-5p targets cyclin T2 (CCNT2), the regulatory subunit of positive transcription elongation factor b, a complex that inhibits OL maturation. Furthermore, CCNT2-specific knockdown promoted rOPC differentiation while not affecting cell cycle status. Together, these data support a dual role for miR-297c-5p as both a negative regulator of OPC proliferation and a positive regulator of OL maturation via its interaction with CCNT2. SIGNIFICANCE STATEMENT: This work describes the role of oligodendrocyte progenitor cell (OPC) microRNAs (miRNAs) during remyelination and development in vivo and differentiation in vitro. This work highlights the importance of miRNAs to OPC biology and describes miR-297c-5p, a novel regulator of OPC function. In addition, we identified CCNT2 as a functional target, thus providing a mechanism by which miR-297c-5p imparts its effects on differentiation. These data are important, given our lack of understanding of OPC miRNA regulatory networks and their potential clinical value. Therefore, efforts to understand the role of miR-297c-5p in pathological conditions and its potential for facilitating repair may provide future therapeutic strategies to treat demyelination.

How to make an oligodendrocyte.

Oligodendrocytes produce myelin, an insulating sheath required for the saltatory conduction of electrical impulses along axons. Oligodendrocyte loss results in demyelination, which leads to impaired neurological function in a broad array of diseases ranging from pediatric leukodystrophies and cerebral palsy, to multiple sclerosis and white matter stroke. Accordingly, replacing lost oligodendrocytes, whether by transplanting oligodendrocyte progenitor cells (OPCs) or by mobilizing endogenous progenitors, holds great promise as a therapeutic strategy for the diseases of central white matter. In this Primer, we describe the molecular events regulating oligodendrocyte development and how our understanding of this process has led to the establishment of methods for producing OPCs and oligodendrocytes from embryonic stem cells and induced pluripotent stem cells, as well as directly from somatic cells. In addition, we will discuss the safety of engrafted stem cell-derived OPCs, as well as approaches by which to modulate their differentiation and myelinogenesis in vivo following transplantation.

Functional consequences of ethidium bromide demyelination of the mouse ventral spinal cord.

Ethidium bromide (EB) has been extensively used in the rat as a model of spinal cord demyelination. However, this lesion has not been addressed in the adult mouse, a model with unlimited genetic potential. Here we characterize behavioral function, inflammation, myelin status and axonal viability following bilateral injection of 0.20 mg/mL ethidium bromide or saline into the ventral white matter (VWM) of female C57Bl/6 mice. EB-induced VWM demyelination significantly reduced spared VWM and Basso Mouse Scale (BMS) scores persisting out to 2 months. Chronic hindlimb dysfunction was accompanied by a persistent inflammatory response (demonstrated by CD45(+) immunofluorescence) and axonal loss (demonstrated by NF-M immunofluorescence and electron microscopy; EM). These cellular responses differ from the rat where inflammation resolves by 3-4 weeks and axon loss is minimal following EB demyelination. As these data suggest that EB-injection in the mouse spinal cord is a non-remyelinating lesion, we sought to ask whether wheel running could promote recovery by enhancing plasticity of local lumbar circuitry independent of remyelination. This did not occur as BMS and Treadscan assessment revealed no significant effect of wheel running on recovery. However, this study defines the importance of descending ventral motor pathways to locomotor function in the mouse as VWM loss results in a chronic hindlimb deficit.

Dorsally-derived oligodendrocytes in the spinal cord contribute to axonal myelination during development and remyelination following focal demyelination.

In the developing spinal cord, the majority of oligodendrocytes are derived from the ventral ventricular zone. Several recent studies suggested that a small number of oligodendrocyte precursor cells (OPCs) can also be generated in the dorsal spinal cord. However, it is not clear whether these dorsal oligodendrocyte precursor cells participate in myelination and remyelination. To investigate the fate and potential function of these dorsally-derived oligodendrocytes (dOLs) in the adult spinal cord, Cre-lox genetic fate mapping in transgenic mice was employed. We used the Pax3(Cre) knock-in mouse to drive Cre expression in the entire dorsal epithelium and the Rosa26-lacZ or Z/EG reporter line to trace their spatial distribution and population dynamics in the spinal cord. The dorsal OPCs generated from the Pax3-expressing domains migrate into all regions of spinal cord and subsequently undergo terminal differentiation and axonal myelination. In response to a focal demyelination injury, a large number of newly differentiated oligodendrocytes originated from dOLs, suggesting that dOLs may provide an important source of OPCs for axonal remyelination in multiple sclerosis or spinal cord injury.

Pyridoxine administration improves behavioral and anatomical outcome after unilateral contusion injury in the rat.

The purpose of this project was to evaluate the preclinical efficacy of pyridoxine, or vitamin B(6). Rats received a 3.0 mm unilateral controlled cortical impact (CCI) injury of the sensorimotor cortex or sham surgery. Treatment with vitamin B(6) (600 or 300 mg/kg IP) or vehicle was administered at 30 min and 24 h post-CCI. Somatosensory dysfunction was evaluated with the vibrissae-forelimb placing and bilateral tactile adhesive removal tests. Sensorimotor dysfunction was evaluated with the locomotor placing and the forelimb asymmetry tests. On the forelimb asymmetry test both treatment groups displayed no asymmetry bias on any of the testing days post-CCI and were statistically no different than the shams. Both vitamin B(6) groups displayed a significant improvement in behavioral performance on the locomotor placing test compared to the vehicle-treated group. Administration of 600 mg/kg also significantly reduced tactile adhesive removal latencies on days 2, 4, 6, and 12 post-CCI. Both treatment groups were improved in their rate of recovery post-CCI on the vibrissae-forelimb placing test, but only the recovery seen in the 600-mg/kg group was significantly improved compared to vehicle. Finally, the 600-mg/kg dose resulted in significant cortical sparing compared to the vehicle-treated group. In general, the effects of vitamin B(6) on recovery of function were dose-dependent, with the 600-mg/kg dose consistently showing greater recovery than the 300-mg/kg dose. More experimental analyses are warranted to evaluate the potential preclinical efficacy and mechanistic action of vitamin B(6).