A ketogenic diet rescues the murine succinic semialdehyde dehydrogenase deficient phenotype

Abstract

Succinic semialdehyde dehydrogenase (SSADH) deficiency is a heritable disorder of GABA degradation characterized by ataxia, psychomotor retardation and seizures. To date, there is no effective treatment for SSADH deficiency. We tested the hypothesis that a ketogenic diet (KD) would improve outcome in an animal model of SSADH deficiency, the SSADH knockout mouse (Aldh5a1-/-). Using a 4:1 ratio of fat to combined carbohydrate and protein KD we set out to compare the general phenotype, in vivo and in vitro electrophysiology and [35S]TBPS binding in both Aldh5a1-/- mice and control (Aldh5a1+/+) mice. We found that the KD prolonged the lifespan of mutant mice by >300% with normalization of ataxia, weight gain and EEG compared to mutants fed a control diet. Aldh5a1-/- mice showed significantly reduced mIPSC frequency in CA1 hippocampal neurons as well as significantly decreased [35S]TBPS binding in all brain areas examined. In KD fed mutants, mIPSC activity normalized and [35S]TBPS binding was restored in the cortex and hippocampus. The KD appears to reverse toward normal the perturbations seen in Aldh5a1-/- mice. Our data suggest that the KD may work in this model by restoring GABAergic inhibition. These data demonstrate a successful experimental treatment for murine SSADH deficiency using a KD, giving promise to the idea that the KD may be successful in the clinical treatment of SSADH deficiency.

Figure 1. Phenotype data (lifespan, ataxia, weights)

This figure outlines the KD’s effects on behavior in SSADH deficient mice. a) CD fed mutants lived an average of 23.78±2.70 days (n=9, range: 18–43 days) whereas KD fed mutants lived 93.50±13.39 days (n=9, range: 58–165 days). Not only did the KD fed mutant mice live longer, but they appeared much healthier— as evidenced by improved weight gain and reduced ataxia. b) All mice were weighed daily from P12 onward. CD fed mutants lost an average of 0.003±0.013g per day (n=9) while KD fed mutants gained 0.13±0.01g per day (n=9). CD fed wildtype mice showed normal weight gain for a mouse, which was significantly higher than both KD and CD fed mutants. The KD is generally associated with stunted growth (both in animals and humans), however, in Aldh5a1^−/− mice it allows for significantly more weight gain than CD. c) Ataxia was assessed daily. CD fed mutants progressed very quickly to high levels of ataxia (n=9). KD fed mutants eventually reached similar levels of ataxia, but took significantly longer to arrive at that stage (n=9). ***p<0.0001, d=days, g=grams.

Figure 2. ECoG Recordings in CD and KD fed Aldh5a1^−/− Mice

a) The baseline ECoG in CD fed Aldh5a1^−/− (n=4) consisted of 35 to 60uV cortical activity at 4 to 7 Hz activity with intermingled 3–5 Hz often interrupted by intermittent 250–300uV bursts of spontaneous, recurrent spike and wave discharges (SWD), which onset/offset was time-locked with absence-like ictal behavior which consisted of frozen immobility, facial myoclonus, and vibrissal twitching. There were also runs of SWD associated with ictal forelimb clonic movements. The baseline of the KD fed Aldh5a1^−/− mice showed a fairly well regulated 35 to 50uV electrical fields at 4 Hz, with decreased interictal or ictal bursts of SWD, recorded at 6, 15 and 30 mm/sec. Time / voltage scale indicate 50uV/ 1 second. Sensitivity= 30uV/mm; LFF= 1 Hz; HHF= 100 Hz; Notched Filter (60 Hz) on; LF= Left Frontal; RF= Right frontal; LP= Left Parietal; RP= Right Parietal. b) The number of convulsions per hour were calculated in CD and KD fed mutants. A convulsion was defined as ictal activity with corresponding clonus, tonus, running or jumping.

Figure 3. Electrophysiology and [³⁵S]TBPS data

a) mIPSCs were recorded in the presence of the voltage gated sodium channel blocker TTX (1µM) and the glutamatergic blockers CNQX (100µM) and APV (20µM). The presence of cesium in the patching electrode blocked GABA_B receptor associated potassium channel conductance. mIPSC activity is dramatically reduced in CD fed Aldh5a1^−/− mice (n=6) compared to CD fed wildtype controls (n=8). KD fed mutants (n=6) had completely restored mIPSC activity. N's indicate the number of animals—recordings were collected from at least two cells from each animal. b) Representative mIPSC traces. c) Representative mEPSC traces. d) mEPSCs were recorded in the presence of the voltage-gated sodium channel blocker TTX (1µM) and the GABAergic blocker BMI (10µM). A similar, albeit non-significant, trend is apparent here as seen with the mIPSCs. mEPSC activity is reduced non-significantly in CD fed mutant mice (n=8). mEPSC frequency is similar in KD fed mutant mice (n=9) and CD fed wildtype mice (n=8). e) For the [³⁵S]TBPS binding we added a KD fed Aldh5a1^+/+ group. Each binding study involved 4 slices per mouse, with n mice per group. The binding experiment was performed in duplicate, meaning (n*4)*2 slices were analyzed. Consistent with previous studies, [³⁵S]TBPS binding was significantly reduced in CD fed Aldh5a1^−/− mice (n=3) compared to CD fed Aldh5a1^+/+ mice (n=5) in all regions. [³⁵S]TBPS binding was completely restored in hippocampus and cortex of KD fed Aldh5a1^−/− mice (n=3). [³⁵S]TBPS binding was partially restored in amydala and thalamus of KD fed mutants. KD fed Aldh5a1^+/+ (n=4) did not differ from CD fed controls. All n’s indicate the number of animals per group. HPC= hippocampus AMY= amygdala THAL= thalamus, *p<0.05 ***p<0.001.