Deletion of Class II ADP-Ribosylation Factors in Mice Causes Tremor by the Nav1.6 Loss in Cerebellar Purkinje Cell Axon Initial Segments

Abstract

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4 ϩ/Ϫ /ARF5 Ϫ/Ϫ mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4 ϩ/Ϫ /ARF5 Ϫ/Ϫ mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4 ϩ/Ϫ /ARF5 Ϫ/Ϫ mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of poreforming voltage-dependent Na ϩ channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4 ϩ/Ϫ / ARF5 Ϫ/Ϫ mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4 ϩ/Ϫ /ARF5 Ϫ/Ϫ mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS.

ARF family GTPases with links to cilia

“…Depletion of either ARF4 or ARF5 from primary keratinocytes inhibited Notch signaling (components of Notch signaling are localized to the centrosome), suggesting that each ARF can affect ciliary function but do not provide insight into the mechanism of such inhibition (40). ARF4 and ARF5 display quite distinct phenotypes when deleted in mice, since mice deleted of ARF5 have normal life expectancy and no defects have been reported in any of their tissues, in contrast to ARF4 null mice, which are embryonic lethal (67). The lack of retinal or kidney dysfunction in ARF5 Ϫ/Ϫ mice argues against ARF5 regulating a key ciliary function on its own, although it may share some relevant activities with ARF4.…”

The Ser19Stop single nucleotide polymorphism (SNP) of human PHYHIPL affects the cerebellum in mice

“…We performed immunohistochemistry as described previously [ 25 ]. C57BL/6 J male mice were anesthetized deeply with a combination of midazolam, medetomidine and butorphanol tartrate and transcardially perfused with PBS and then with Zamboni's fixative (2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, containing 0.2% picric acid).…”