Muscarinic Receptor M3R Signaling Prevents Efficient Remyelination by Human and Mouse Oligodendrocyte Progenitor Cells.

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 M3R is the functionally relevant receptor. Lentiviral M3R knockdown in human primary CD140a/PDGFαR+ OPCs resulted in enhanced differentiation in vitro and substantially reduced the calcium response following muscarinic agonist treatment. Importantly, following transplantation in hypomyelinating shiverer/rag2 mice, M3R knockdown improved remyelination by human OPCs. Furthermore, conditional M3R ablation in adult NG2-expressing OPCs increased oligodendrocyte differentiation and led to improved spontaneous remyelination in mice. Together, we demonstrate that M3R receptor mediates muscarinic signaling in human OPCs that act to delay differentiation and remyelination, suggesting that M3 receptors are viable targets for human demyelinating disease.SIGNIFICANCE STATEMENT 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, we show that genetic M3R ablation in both mouse and human cells results in improved remyelination and is mediated by acceleration of oligodendrocyte commitment from oligodendrocyte progenitor cells. Therefore, M3R represents an attractive target for induced remyelination in human disease.

KCNQ2/3 regulates efferent mediated slow excitation of vestibular afferents in mammals.

Primary vestibular afferents transmit information from hair cells about head position and movement to the CNS, which is critical for maintaining balance, gaze stability and spatial navigation. The CNS, in turn, modulates hair cells and afferents via the efferent vestibular system (EVS) and its activation of several cholinergic signaling mechanisms. Electrical stimulation of EVS neurons gives rise to three kinetically- and mechanistically-distinct afferent responses including a slow excitation, a fast excitation, and a fast inhibition. EVS-mediated slow excitation is attributed to odd-numbered muscarinic acetylcholine receptors (mAChRs) on the afferent whose activation leads to the closure of a potassium conductance and increased afferent discharge. Likely effector candidates include low-threshold, voltage-gated potassium channels belonging to the KCNQ (Kv7.X) family, which are involved in neuronal excitability across the nervous system and are subject to mAChR modulation. Specifically, KCNQ2/3 heteromeric channels may be the molecular correlates for the M-current, a potassium current that is blocked following the activation of odd-numbered mAChRs. To this end, multiple members of the KCNQ channel family, including KCNQ2 and KCNQ3, are localized to several microdomains within vestibular afferent endings, where they influence afferent excitability and could be targeted by EVS neurons. Additionally, the relative expression of KCNQ subunits appears to vary across the sensory epithelia and among different afferent types. However, it is unclear which KCNQ channel subunits are targeted by mAChR activation and whether that also varies among different afferent classes. Here we show that EVS-mediated slow excitation is blocked and enhanced by the non-selective KCNQ channel blocker XE991 and opener retigabine, respectively. Using KCNQ subunit-selective drugs, we observed that a KCNQ2 blocker blocks the slow response in irregular afferents, while a KCNQ2/3 opener enhances slow responses in regular afferents. The KCNQ2 blockers did not appear to affect resting afferent discharge rates, while KCNQ2/3 or KCNQ2/4 openers decreased afferent excitability. Here, we show pharmacological evidence that KCNQ2/3 subunits are likely targeted by mAChR activation in mammalian vestibular afferents. Additionally, we show that KCNQ3 KO mice have altered resting discharge rate as well as EVS-mediated slow response. These data together suggest that KCNQ channels play a role in slow response and discharge rate of vestibular afferents, which can be modulated by EVS in mammals.

Elucidating the role of muscarinic acetylcholine receptor (mAChR) signaling in efferent mediated responses of vestibular afferents in mammals.

The peripheral vestibular system detects head position and movement through activation of vestibular hair cells (HCs) in vestibular end organs. HCs transmit this information to the CNS by way of primary vestibular afferent neurons. The CNS, in turn, modulates HCs and afferents via the efferent vestibular system (EVS) through activation of cholinergic signaling mechanisms. In mice, we previously demonstrated that activation of muscarinic acetylcholine receptors (mAChRs), during EVS stimulation, gives rise to a slow excitation that takes seconds to peak and tens of seconds to decay back to baseline. This slow excitation is mimicked by muscarine and ablated by the non-selective mAChR blockers scopolamine, atropine, and glycopyrrolate. While five distinct mAChRs (M1-M5) exist, the subtype(s) driving EVS-mediated slow excitation remain unidentified and details on how these mAChRs alter vestibular function is not well understood. The objective of this study is to characterize which mAChR subtypes drive the EVS-mediated slow excitation, and how their activation impacts vestibular physiology and behavior. In C57Bl/6J mice, M3mAChR antagonists were more potent at blocking slow excitation than M1mAChR antagonists, while M2/M4 blockers were ineffective. While unchanged in M2/M4mAChR double KO mice, EVS-mediated slow excitation in M3 mAChR-KO animals were reduced or absent in irregular afferents but appeared unchanged in regular afferents. In agreement, vestibular sensory-evoked potentials (VsEP), known to be predominantly generated from irregular afferents, were significantly less enhanced by mAChR activation in M3mAChR-KO mice compared to controls. Finally, M3mAChR-KO mice display distinct behavioral phenotypes in open field activity, and thermal profiles, and balance beam and forced swim test. M3mAChRs mediate efferent-mediated slow excitation in irregular afferents, while M1mAChRs may drive the same process in regular afferents.

The mammalian efferent vestibular system utilizes cholinergic mechanisms to excite primary vestibular afferents.

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 DHßE selectively blocks efferent-mediated fast excitation, while leaving the efferent-mediated slow component intact. These data confirm that mammalian EVS actions are predominantly cholinergic.

Characterizing the Access of Cholinergic Antagonists to Efferent Synapses in the Inner Ear.

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 α4ß2*-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.

Paired Related Homeobox Protein 1 Regulates Quiescence in Human Oligodendrocyte Progenitors.

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.