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

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.

Movie 2. Home cage behavior of an FSDS mouse.This movie illustrates the behavior of an FSDS mouse.In contrast to a normal FVB/N mouse, these mice remain immobile for long periods of time and do not explore their surroundings.
Movie 3. Home cage behavior of mouse following pilocarpine-induced status epilepticus.This movie illustrates the behavior of a mouse which has undergone status epilepticus following injection with pilocarpine.As with FSDS mice, these mice remain immobile for long periods of time and also do not explore their surroundings.
Movie 4. Spontaneous seizure observed in an FSDS mouse.This movie shows an FSDS mouse undergoing a spontaneous seizure.A normal FVB/N littermate walks past the seizing mouse.
Movie 5. Auditory SUDEP in an FSDS mouse.This movie shows an FSDS mouse undergoing SUDEP in response to an auditory stimulus.Before the auditory stimulus, the FSDS mouse is immobile.When the sound begins, the mouse responds by moving about the cage, first slowly then in a more frantic manner.Finally, the mouse exhibits a "popcorn" seizure and dies.
Movie 6.Comparison of normal FVB/N mouse and FSDS mouse in response to auditory stimulus.This movie the response of a normal FVB/N mouse and an FSDS mouse to an auditory stimulus.Before the sound begins, the normal FVB/N mouse is exploring the cage while the FSDS mouse is immobile.The auditory stimulus has no impact on the normal FVB/N mouse's behavior.However, the auditory stimulus causes the FSDS mouse to move frantically about the cage, seize and then die.
Figure S3 and S4 show EEG recordings from two FSDS mice.The top tracing displays a 130-second epoch showing a burst of high-amplitude spike discharges.Underneath is a close-up view of the spike burst showing synchronous epileptiform discharges from the 3 EEG channels along with concurrent trace from accelerometer x-axis.Each trace is 50 seconds in duration.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).Supplementary Figure 5. Electroencephalogram (EEG) traces from normal WT FVB/N mice do not show any seizure activity -Example 1. Figure S5-S10 represent recordings obtained from one of the six normal FVB/N mice included in this analysis.Each trace is 50 seconds in duration.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).Supplementary Figure 6.Electroencephalogram (EEG) traces from normal WT FVB/N mice do not show any seizure activity -Example 2. Supplementary Figure 7. Electroencephalogram (EEG) traces from normal WT FVB/N mice do not show any seizure activity -Example 3. Supplementary Figure 8. Electroencephalogram (EEG) traces from normal WT FVB/N mice do not show any seizure activity -Example 4. Supplementary Figure 9. Electroencephalogram (EEG) traces from normal WT FVB/N mice do not show any seizure activity -Example 5. Supplementary Figure 10.Electroencephalogram (EEG) traces from normal WT FVB/N mice do not show any seizure activity -Example 6. Supplementary Figure 11.Mouse models of epilepsy exhibit astrocytosis and upregulation of BDNF expression.In order to examine the seizure induced neurodegeneration in FSDS mice, we compared the brains of these mice with two established chemical models of epilepsy: kindling with penteylenetetrazole (PTZ) and pilocarpine induced status epilepticus.(A) As with FSDS mice, both pilocarpine and PTZ treated mice exhibited extensive astrocytosis in the hippocampus and piriform cortex.(B) Similarly, both of these chemical models of epilepsy showed increased levels of BDNF protein.(C) Quantification of BDNF levels showed that the increase in BDNF expression in FSDS mice was similar or greater to that in the two chemically induced seizure models (N=2 FSDS, N=2 Normal FVB/N, N=2 FVB/N mice pilocarpine-induced status epilepticus, N=3 FVB/N mice with PTZ kindling).Error bars indicates standard error of the mean.Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparisons test.** p < 0.01.Supplementary Figure12.FSDS incidence is unaltered by handling-stress, inflammation or auditory stimuli.Having shown that a decrease in Htt levels increased the frequency of the FSDS phenotype, we sought to determine whether the proportion of mice that develop this phenotype could be increased by manipulations that have previously been shown to influence the development of epilepsy in mouse or rat models.Unlike decreasing levels of Htt, the frequency of the FSDS phenotype was not altered by repeated handling, induction of inflammation with LPS (lipopolysaccharide) or auditory stimulation.Statistical significance was assessed using the Chi-squared test.** p<0.01.

Table 1 .
Summary of statistical analyses.Home cage behavior of a normal FVB/N mouse.This movie illustrates the behavior of a normal FVB/N mouse.Notice that the mouse actively explores the cage.