SUMMARY Human oligodendrocyte progenitor cells (hOPCs) persist into adulthood as an abundant precursor population capable of division and differentiation. The transcriptional mechanisms that regulate hOPC homeostasis remain poorly defined. Herein, we identify paired related homeobox protein 1 (PRRX1) in primary PDGFαR+ hOPCs. We show that enforced PRRX1 expression results in reversible G1/0 arrest. While both PRRX1 splice variants reduce hOPC proliferation, only PRRX1a abrogates migration. hOPC engraftment into hypomyelinated shiverer/rag2 mouse brain is severely impaired by PRRX1a, characterized by reduced cell proliferation and migration. PRRX1 induces a gene expression signature characteristic of stem cell quiescence. Both IFN-γ and BMP signaling upregulate PRRX1 and induce quiescence. PRRX1 knockdown modulates IFN-γ-induced quiescence. In mouse brain, PRRX1 mRNA was detected in non-dividing OPCs and is upregulated in OPCs following demyelination. Together, these data identify PRRX1 as a regulator of quiescence in hOPCs and as a potential regulator of pathological quiescence.

Electrical stimulation of the mammalian efferent vestibular system (EVS) predominantly excites primary vestibular afferents along two distinct time scales. Although roles for acetylcholine (ACh) have been demonstrated in other vertebrates, synaptic mechanisms underlying mammalian EVS actions are not well-characterized. To determine if activation of ACh receptors account for efferent-mediated afferent excitation in mammals, we recorded afferent activity from the superior vestibular nerve of anesthetized C57BL/6 mice while stimulating EVS neurons in the brainstem, before and after administration of cholinergic antagonists. Using a normalized coefficient of variation (CV*), we broadly classified vestibular afferents as regularly- (CV* < 0.1) or irregularly-discharging (CV* > 0.1) and characterized their responses to midline or ipsilateral EVS stimulation. Afferent responses to efferent stimulation were predominantly excitatory, grew in amplitude with increasing CV*, and consisted of fast and slow components that could be identified by differences in rise time and post-stimulus duration. Both efferent-mediated excitatory components were larger in irregular afferents with ipsilateral EVS stimulation. Our pharmacological data show, for the first time in mammals, that muscarinic AChR antagonists block efferent-mediated slow excitation whereas the nicotinic AChR antagonist DHβE selectively blocks efferent-mediated fast excitation, while leaving the efferent-mediated slow component intact. These data confirm that mammalian EVS actions are predominantly cholinergic.

Muscarinic receptor antagonists act as potent inducers of oligodendrocyte differentiation and accelerate remyelination. However, the use of muscarinic antagonists in the clinic is limited by poor understanding of the operant receptor subtype, and questions regarding possible species differences between rodents and humans. Based on high selective expression in human oligodendrocyte progenitor cells (OPCs), we hypothesized that MR is the functionally relevant receptor. Lentiviral MR knock-down in human primary CD140a/PDGFαR OPCs resulted in enhanced differentiation and substantially reduced the calcium response following muscarinic agonist treatment. Importantly, following transplantation in hypomyelinating mice, MR knock-down improved remyelination by human OPCs. Furthermore, conditional MR ablation in adult NG2-expressing OPCs increased oligodendrocyte differentiation and led to improved spontaneous remyelination in mice. Together, we demonstrate that MR receptor mediates muscarinic signaling in human OPCs that act to delay differentiation and remyelination, suggesting that M receptors are viable targets for human demyelinating disease.The identification of drug targets aimed at improving remyelination in patients with demyelination disease is a key step in development of effective regenerative therapies to treat diseases such as multiple sclerosis. Muscarinic receptor antagonists have been identified as effective potentiators of remyelination but the receptor subtypes that mediate these receptors are unclear. In this study, Welliver et al. show that genetic MR ablation in both mouse and human cells results in improved remyelination and is mediated by acceleration of oligodendrocyte commitment from oligodendrocyte progenitor cells. Therefore, MR therefore represents an attractive target for induced remyelination in human disease.

Stimulation of cholinergic efferent neurons innervating the inner ear has profound, well-characterized effects on vestibular and auditory physiology, after activating distinct ACh receptors (AChRs) on afferents and hair cells in peripheral endorgans. Efferent-mediated fast and slow excitation of vestibular afferents are mediated by α4β2*-containing nicotinic AChRs (nAChRs) and muscarinic AChRs (mAChRs), respectively. On the auditory side, efferent-mediated suppression of distortion product otoacoustic emissions (DPOAEs) is mediated by α9α10nAChRs. Previous characterization of these synaptic mechanisms utilized cholinergic drugs, that when systemically administered, also reach the CNS, which may limit their utility in probing efferent function without also considering central effects. Use of peripherally-acting cholinergic drugs with local application strategies may be useful, but this approach has remained relatively unexplored. Using multiple administration routes, we performed a combination of vestibular afferent and DPOAE recordings during efferent stimulation in mouse and turtle to determine whether charged mAChR or α9α10nAChR antagonists, with little CNS entry, can still engage efferent synaptic targets in the inner ear. The charged mAChR antagonists glycopyrrolate and methscopolamine blocked efferent-mediated slow excitation of mouse vestibular afferents following intraperitoneal, middle ear, or direct perilymphatic administration. Both mAChR antagonists were effective when delivered to the middle ear, contralateral to the side of afferent recordings, suggesting they gain vascular access after first entering the perilymphatic compartment. In contrast, charged α9α10nAChR antagonists blocked efferent-mediated suppression of DPOAEs only upon direct perilymphatic application, but failed to reach efferent synapses when systemically administered. These data show that efferent mechanisms are viable targets for further characterizing drug access in the inner ear.

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