Hcn1 is a tremorgenic genetic component in a rat model of essential tremor

Abstract

Genetic factors are thought to play a major role in the etiology of essential tremor (ET); however, few genetic changes that induce ET have been identified to date. In the present study, to find genes responsible for the development of ET, we employed a rat model system consisting of a tremulous mutant strain, TRM/Kyo (TRM), and its substrain TRMR/Kyo (TRMR). The TRM rat is homozygous for the tremor (tm) mutation and shows spontaneous tremors resembling human ET. The TRMR rat also carries a homozygous tm mutation but shows no tremor, leading us to hypothesize that TRM rats carry one or more genes implicated in the development of ET in addition to the tm mutation. We used a positional cloning approach and found a missense mutation (c. 1061 C>T, p. A354V) in the hyperpolarization-activated cyclic nucleotide-gated 1 channel (Hcn1) gene. The A354V HCN1 failed to conduct hyperpolarization-activated currents in vitro, implicating it as a loss-of-function mutation. Blocking HCN1 channels with ZD7288 in vivo evoked kinetic tremors in nontremulous TRMR rats. We also found neuronal activation of the inferior olive (IO) in both ZD7288-treated TRMR and non-treated TRM rats and a reduced incidence of tremor in the IO-lesioned TRM rats, suggesting a critical role of the IO in tremorgenesis. A rat strain carrying the A354V mutation alone on a genetic background identical to that of the TRM rats showed no tremor. Together, these data indicate that body tremors emerge when the two mutant loci, tm and Hcn1A354V, are combined in a rat model of ET. In this model, HCN1 channels play an important role in the tremorgenesis of ET. We propose that oligogenic, most probably digenic, inheritance is responsible for the genetic heterogeneity of ET.

Fig 1. TRM rat as a model of essential tremor.

A: Representative EMG from TRM rats. Tremor is shown as a bold line above EMG. Lower panels show magnified EMG and its power frequency analysis (red bar). Calibration: 100 μV and 20 s (upper panel), 50 μV and 5 s (lower panel). B: Effects of anti-tremor agents on tremor incidence in TRM rats. Data are presented as the mean ± SEM of seven (propranolol and trihexyphenidyl) or six (phenobarbital) animals. *P<0.05, **P<0.01, vs. pre-drug control levels (pre).

Fig 2. Identification of trm2 mutation.

A: Distribution of haplotypes around trm2 in (TRM × TRMR)F1 × TRM backcross progeny. White boxes, animals heterozygous for TRM alleles. Black boxes, animals homozygous for TRM alleles. Number of backcross progeny specified underneath the haplotypes. B: Linkage and physical maps including trm2. Parp8, poly (ADP-ribose) polymerase family, member 8; Emb, embigin; Mrps30, mitochondrial ribosomal protein S30; Fgf10, fibroblast growth factor 10. C: Sequencing analysis in TRMR (upper) and TRM (lower) rats. A nucleotide change from C to T located in Hcn1 exon 4 is indicated by arrow. The mutation results in an amino acid substitution of alanine (Ala) with valine (Val) at codon 354 of the HCN1 protein. D: Schematic representation of the HCN1 channel subunit. P-loop, pore region; CNBD, cyclic nucleotide-binding domain; black circle, A354V substitution located in the pore region. E: Representative current recordings of wild-type and A354V HCN1 channels. Right panel, hyperpolarization-induced currents measured at the end of the step pulse (-120 mV). Data are presented as the mean ± SEM of seven (wild-type) or six (A354V) experiments. **P<0.01 vs. wild-type.

Fig 3. Immunohistochemical analysis of Fos expression in TRM rats.

A: Schematic illustrations of brain sections selected for quantitative analysis of Fos-immunoreactivity. Anteroposterior distance from bregma is shown above each brain section. Filled boxes in each section indicate the areas analyzed. B: Numbers of Fos-immunoreactive neurons in various brain regions. Data show the mean ± SEM of four (TRM) or five (WTC) rats. *P<0.05, **P<0.01 vs. control (WTC).

Fig 4. Tremor induction by the HCN1 channel blocker ZD7288 in TRMR rats.

A: Genetic components responsible for tremor development in our rat model of ET. TRM rats, carrying both the tm deletion (red) and the Hcn1 mutation (blue), showed body tremors. TRMR rats, carrying the tm deletion but not the Hcn1 mutation, showed no body tremors, but body tremors were induced when the selective HCN1 channel blocker ZD7288 was administered (see B, this figure). WTC rats, carrying the Hcn1 mutation but not the tm deletion, showed no body tremors with or without administration of ZD7288 (see B, this figure). B: Effects of ZD7288 on tremor induction in nontremulous TRMR rats. Duration and intensity of tremor observed in TRMR rats that received vehicle or ZD7288. Data are presented as the mean ± SEM of seven (vehicle) or eight (ZD7288) animals. *P<0.05, **P<0.01 vs. pre-treatment control levels (pre).

Fig 5. Immunohistochemical analysis of Fos expression induced by ZD7288 in TRMR rats.

Effects of ZD7288 on regional Fos expression in TRMR rats. Inset: representative photographs of Fos-immunoreactive cells in the IO. Scale bar: 100 μm. Data are presented as the mean ± SEM of seven (vehicle) or eight (ZD7288) animals. *P<0.05 vs. control (vehicle).

Fig 6. Tremor inhibition by IO lesioning in TRM rats.

A: Effects of IO lesioning on tremor induction in TRM rats. The duration and intensity of tremor was significantly suppressed 38 and 54 h after IO lesioning. Data are presented as the mean ± SEM of five animals. *P<0.05 vs. pre-treatment control. B: Lesion sites in the brain sections.