Huntingtin Decreases Susceptibility to a Spontaneous Seizure Disorder in FVN/B Mice

Abstract

Huntington disease (HD) is an adult-onset neurodegenerative disorder that is caused by a trinucleotide CAG repeat expansion in the HTT gene that codes for the protein huntingtin (HTT in humans or Htt in mice). HTT is a multi-functional, ubiquitously expressed protein that is essential for embryonic survival, normal neurodevelopment, and adult brain function. The ability of wild-type HTT to protect neurons against various forms of death raises the possibility that loss of normal HTT function may worsen disease progression in HD. Huntingtin-lowering therapeutics are being evaluated in clinical trials for HD, but concerns have been raised that decreasing wild-type HTT levels may have adverse effects. Here we show that Htt levels modulate the occurrence of an idiopathic seizure disorder that spontaneously occurs in approximately 28% of FVB/N mice, which we have called FVB/N Seizure Disorder with SUDEP (FSDS). These abnormal FVB/N mice demonstrate the cardinal features of mouse models of epilepsy including spontaneous seizures, astrocytosis, neuronal hypertrophy, upregulation of brain-derived neurotrophic factor (BDNF), and sudden seizure-related death. Interestingly, mice heterozygous for the targeted inactivation of Htt (Htt+/- mice) exhibit an increased frequency of this disorder (71% FSDS phenotype), while over-expression of either full length wild-type HTT in YAC18 mice or full length mutant HTT in YAC128 mice completely prevents it (0% FSDS phenotype). Examination of the mechanism underlying huntingtin's ability to modulate the frequency of this seizure disorder indicated that over-expression of full length HTT can promote neuronal survival following seizures. Overall, our results demonstrate a protective role for huntingtin in this form of epilepsy and provide a plausible explanation for the observation of seizures in the juvenile form of HD, Lopes-Maciel-Rodan syndrome, and Wolf-Hirschhorn syndrome. Adverse effects caused by decreasing huntingtin levels have ramifications for huntingtin-lowering therapies that are being developed to treat HD.

Figure 1.

FSDS mice exhibit abnormal activity and megacephaly. (A) Normal FVB/N mice (N = 28) exhibited a uniform pattern of activity where activity declines over the one-hour open field trial (each line represents one mouse). (B) In contrast, FSDS mice (N = 31) exhibited multiple periods of both hypoactivity and hyperactivity. (C) The brains of FSDS mice (shown with # sign) were much larger than the brains of normal FVB/N mice. (D) FSDS mice exhibited a 46% increase in brain weight resulting only from an increase in the forebrain, while the cerebellum is spared (N = 12 WT, 4 FSDS). (E-F) The brains of FSDS mice also exhibited a significant increase in brain cross-sectional area compared to normal FVB/N mice (N = 6 WT, 5 FSDS). Statistical significance in panel D was determined using a two-way ANOVA with Šidák’s multiple comparisons test. Statistical significance in panel F was determined using the Mann-Whitney test. Error bars indicate SEM. ** p<0.01, **** p<0.0001.

Figure 2.

Representative EEG from FSDS mice showing epileptiform discharges. (A) Condensed view of 145-seconds epoch showing a burst of high-amplitude discharges. (B) Expanded views of the burst showing synchronous ictal discharges from the 3 EEG channels along with concurrent trace from accelerometer x-axis. The onset of the discharges coincides with changes in the accelerometer that becomes more intense with time and then tapers down and flattens towards the end of the episode. (C) Further expanded views showing waveform morphology of post-ictal spikes (i) and in 8-seconds samples taken form the beginning (ii), middle (iii) and end (iv) of the epoch. Abbreviations, FCx-L: left frontal cortex, FCx-R: right frontal cortex, PCx: parietal cortex, sec: seconds, mV: millivolts, G: acceleration of Earth’s gravity (~9.8 m/s).

Figure 3.

Brains from FSDS mice exhibit neuropathology characteristic of mouse models of epilepsy. (A) Immunostaining with a Cy3-labelled anti-GFAP antibody reveals that FSDS mice exhibit astrocytosis selectively in hippocampus, piriform cortex and amygdala (large frame) but not the striatum, while minimal glial fibrillary acidic protein staining is observed in normal FVB/N mice (small inset frame). (B, C) FSDS mice also showed neuronal hypertrophy selectively in piriform cortex and amygdala but not the striatum (N = 3 per group). (D) At the molecular level, FSDS mice showed a dramatic increase in brain-derived neurotrophic factor (BDNF) expression by Western blotting. (E) The increase in BDNF levels was only observed in forebrain regions - not the cerebellum (N = 3 per group). (F) Epigenetic changes at BDNF promoter 2 (dimethylation of H3-K4, acetylation of H4) contribute to the increased BDNF expression in FSDS mice (N = 3 per group). Statistical significance in panels C, E and F was determined using a two-way ANOVA with Šidák’s multiple comparisons test. Error bars indicate SEM. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 4.

Huntingtin expression modulates the frequency of the FSDS epileptic phenotype. (A) To examine the effect of Htt expression on the development of the FSDS phenotype, we compared the FSDS frequency between mice with altered levels of Htt and their wild-type littermates. FSDS mice were identified based on home cage immobility, an aggressive response to handling and megencephaly post-mortem. (B) A 50% reduction in huntingtin (Htt) levels in Htt+/- mice more than doubled the frequency of the FSDS phenotype. (C) Conversely, increasing the expression of full length wild-type HTT completely eliminated the occurrence of the FSDS phenotype in YAC18 mice. (D) Similarly, increased expression of full-length mutant HTT completely prevented the development of the FSDS phenotype in YAC128 mice. (E) In contrast, expression of an N-terminal fragment of mutant HTT in shortstop mice had no effect on the frequency of the FSDS phenotype. The data and statistical analyses for panel A are presented in Table 1. Statistical significance was determined using the Chi Square test. Note that is panels B, D and E littermate controls were used for WT animals. Panel C uses a composite of WT animals from B, D, and E. Htt-Tg = transgenic mice expressing increased levels of full length or mutant HTT. Htt-KO = heterozygote Htt knockout mice.

Figure 5.

Huntingtin levels are unaltered in FSDS mice. Since the levels of full-length Htt clearly modulate the frequency of development of the FSDS phenotype, we sought to determine whether FSDS mice have reduced levels of Htt compared to normal FVB/N mice. (A,B) Western blotting and quantification of Htt levels revealed no difference between normal FVB/N mice (N=9) and FSDS mice (N=7). This indicates that while decreased levels of Htt increase the frequency of the FSDS phenotype, mice that develop the FSDS phenotype do not have decreased levels of Htt. Huntingtin levels were expressed as a ratio of huntingtin over actin. Statistical significance was assessed using the Mann-Whitney test.

Figure 6.

Over-expression of huntingtin reduces seizure-induced neurodegeneration. To assess seizure susceptibility, WT mice (N =17) and YAC18 mice (N=19) were given repeated injections of pentylenetetrazole (PTZ). WT mice (N=7) were also injected with saline as a control. Over-expression of huntingtin (HTT) had no effect on either the maximum seizure severity (A) or the total number of seizures (B). To assess seizure-induced neurodegeneration, WT mice (N=12) and YAC18 mice (N = 9) were injected with pilocarpine. Sections containing the hippocampus were stained for degenerating neurons using fluorojade. Among those mice that developed status epilepticus (6 WT, 4 YAC18), mice over-expressing HTT showed significantly decreased numbers of degenerating neurons (C,D). Statistical significance was assessed using a mixed-effect analysis with Tukey’s multiple comparisons test in panels A and B, and the Mann-Whitney test in panel D. Error bars indicate SEM. * p<0.05.

Figure 7.

Huntingtin protects against epilepsy. A proportion of FVB/N mice develop an idiopathic seizure disorder characterized by megacephaly with features of epilepsy including poct-ictal behaviour, abnormal activity, spontaneous seizures, SUDEP (sudden unexpected death in epilepsy), neuronal hypertrophy, astrocytosis and upregulation of BDNF. We have named these mice FSDS mice (FVB/N Seizure Disorder with SUDEP). The frequency of the FSDS phenotype is modulated by the levels of full-length huntingtin (Htt). Htt+/- mice with 50% decreased levels of Htt have more than double the frequency of FSDS phenotype, while over-expression of HTT in YAC18 mice completely prevents the occurrence of the FSDS phenotype. Examination of the mechanism by which Htt protects against this epilepsy disorder reveals that Htt reduces seizure induced damage but does not affect the number or severity of chemically induced seizures. These results suggest that reducing seizure-induced neuronal damage can limit the subsequent development of epilepsy.