Differential effects of mTOR inhibition and dietary ketosis in a mouse model of subacute necrotizing encephalomyelopathy

Genetic mitochondrial diseases are the most frequent cause of inherited metabolic disorders and one of the most prevalent causes of heritable neurological disease. Leigh syndrome is the most common clinical presentation of pediatric mitochondrial disease, typically appearing in the first few years of life, and involving severe multisystem pathologies. Clinical care for Leigh syndrome patients is difficult, complicated by the wide range of symptoms including characteristic progressive CNS lesion, metabolic sequelae, and epileptic seizures, which can be intractable to standard management. While no proven therapies yet exist for the underlying mitochondrial disease, a ketogenic diet has led to some reports of success in managing mitochondrial epilepsies, with ketosis reducing seizure risk and severity. The impact of ketosis on other aspects of disease progression in Leigh syndrome has not been studied, however, and a rigorous study of the impact of ketosis on seizures in mitochondrial disease is lacking. Conversely, preclinical efforts have identified the intracellular nutrient signaling regulator mTOR as a promising therapeutic target, with data suggesting the benefits are mediated by metabolic changes. mTOR inhibition alleviates epilepsies arising from defects in TSC, an mTOR regulator, but the therapeutic potential of mTOR inhibition in seizures related to primary mitochondrial dysfunction is unknown. Given that ketogenic diet is used clinically in the setting of mitochondrial disease, and mTOR inhibition is in clinical trials for intractable pediatric epilepsies of diverse causal origins, a direct experimental assessment of their effects is imperative. Here, we define the impact of dietary ketosis on survival and CNS disease in the Ndufs4(KO) mouse model of Leigh syndrome and the therapeutic potential of both dietary ketosis and mTOR inhibition on seizures in this model. These data provide timely insight into two important clinical interventions

Region-Specific Defects of Respiratory Capacities in the Ndufs4(KO) Mouse Brain.

Lack of NDUFS4, a subunit of mitochondrial complex I (NADH:ubiquinone oxidoreductase), causes Leigh syndrome (LS), a progressive encephalomyopathy. Knocking out Ndufs4, either systemically or in brain only, elicits LS in mice. In patients as well as in KO mice distinct regions of the brain degenerate while surrounding tissue survives despite systemic complex I dysfunction. For the understanding of disease etiology and ultimately for the development of rationale treatments for LS, it appears important to uncover the mechanisms that govern focal neurodegeneration.Here we used the Ndufs4(KO) mouse to investigate whether regional and temporal differences in respiratory capacity of the brain could be correlated with neurodegeneration. In the KO the respiratory capacity of synaptosomes from the degeneration prone regions olfactory bulb, brainstem and cerebellum was significantly decreased. The difference was measurable even before the onset of neurological symptoms. Furthermore, neither compensating nor exacerbating changes in glycolytic capacity of the synaptosomes were found. By contrast, the KO retained near normal levels of synaptosomal respiration in the degeneration-resistant/resilient "rest" of the brain. We also investigated non-synaptic mitochondria. The KO expectedly had diminished capacity for oxidative phosphorylation (state 3 respiration) with complex I dependent substrate combinations pyruvate/malate and glutamate/malate but surprisingly had normal activity with α-ketoglutarate/malate. No correlation between oxidative phosphorylation (pyruvate/malate driven state 3 respiration) and neurodegeneration was found: Notably, state 3 remained constant in the KO while in controls it tended to increase with time leading to significant differences between the genotypes in older mice in both vulnerable and resilient brain regions. Neither regional ROS damage, measured as HNE-modified protein, nor regional complex I stability, assessed by blue native gels, could explain regional neurodegeneration.Our data suggests that locally insufficient respiration capacity of the nerve terminals may drive focal neurodegeneration

Stability of complex I is not increased in KO mitochondria from resilient "rest" brain.

<p>Blue Native PAGE of mitochondrial proteins from brainstem (BS) and “rest”brain (R) were electrophoresed under conditions which leave wildtype mitochondrial supercomplexes intact (A). Purpelish diaphorase activity staining revealed bands containing the NAD binding site of complex I (black arrow heads). All proteins were nonspecifically counterstained aqua blue with Coomassie Blue. Select landmark bands are labeled: supercomplexes containing complex I III and IV (I III IV), incomplete supercomplex (I III), solitary complex I (I), ATP synthetase (V), a low molecular weight fragment of complex I (I fgmt). (Strong diaphorase activity at grey arrow heads may be dihydrolipoamid DH). Densitometry results (B): Activity stain for the indicated complex I containing bands was normalized against the protein stain of the complex V band (serving as an internal loading control).</p

Capacity for oxidative phosphorylation (state 3 respiration) of isolated non-synaptic mitochondria by brain region and age group.

<p>A: Complex I-dependent state 3 respiration with pyruvate plus malate remained constant with age in the KO but tended to increase in the controls leading to significant capacity shortfalls in older KO mice in all brain regions tested: brain stem (BS), cerebellum (CB) and remainder of the brain excluding olfactory bulb (R). B: With complex I dependent electron transport blocked by rotenone, the capacity for state 3 respiration via complex II increased with age in the controls reaching significance in CB and R. In the KO capacity did not significantly increase with age but was already elevated in the young cohort. The number of biological replicates is stated inside the bars. * denotes significant changes at significance level (α = 5%) after Holm-Bonferroni correction for multiple comparisons.</p

Complex I dependent capacity for oxidative phosphorylation of isolated (non-synaptic) mitochondria.

<p>(A) Maximal ADP-stimulated respiration (state 3) of mitochondria from whole brain fuelled with complex I dependent electron donor substrates. Note, while increase of oxphos capacity with age only reached statistical significance for controls powered with glutamate/malate, it becomes significant (3-way ANOVA P<0.001) for the aggregate of all substrates and all genotypes. (B,C,D) State 3 respiration for mitochondria isolated from brain stem (BS), cerebellum (CB) and remainder of brain (R). The complex I dependent electron donor substrates were pyruvate + malate (B), glutamate + malate (C) and α-ketoglutarate + malate (D). Olfactory bulb was excluded from both whole brain and R preparations. * denotes significant (α = 0.05) differences after Holm's correction for multiple pairwise comparisons.</p

HNE damage to mitochondrial protein from vulnerable regions in KO is not elevated compared to unaffected controls.

<p>Western blots with mitochondrial protein were probed for a covalent modification caused by HNE, a reactive product of ROS-triggered lipid peroxidation. Overall damage was assessed by densitometry of whole lanes. Data were normalized to reference samples which were present on each blot. Note that damage in the degeneration prone regions brainstem (BS) and cerebellum (CB) of the KO does not exceed the damage in the respective regions from controls. * denotes significant difference after Holm Bonferroni correction at the α = 5% level.</p

Metabolic capacity of synaptosomes.

<p>A: Synaptosomes were fuelled with glucose plus pyruvate and mitochondrial respiration was maximized by uncoupling with the protonophore FCCP. Oxygen consumption rates (OCR) were corrected for non-mitochondrial oxygen consumption. Note, the degeneration-prone regions of the KO: olfactory bulb (OB), brainstem (BS) and cerebellum (CB),—but not the resilient “rest” of the brain (R) -, have significantly lower capacities to respire than those of the controls. B: Glycolytic capacity was assessed as the external acidification rate (ECAR) measured after blocking oxidative phosphorylation and stimulating ATP turnover with 4-aminopyridine. Note, for any given brain region the capacity for glycolysis was fairly stable between genotypes and with age. The only significant (α = 5%) change observed was a decrease with age in the controls (*) from cerebellum. The number of biological repeats is stated inside the bars. * denotes significant (α = 5%) differences after Holm-Bonferroni correction for multiple comparisons.</p

Markers for synaptosomes and mitochondria in synaptosomal preparations.

<p>Western blots were simultaneously probed for synaptophysin (red) and the ATPase-α (green). A blot with 3 independent brainstem (BS) synaptosome preparations for each combination of genotype and age group is shown as an example (A). 14 additional blots (not shown) were evaluated to cover samples from this and the other brain regions. The same “rest” brain (R) synaptosome preparation (Reference) was present on each blot for normalization. Densitometry results for synaptophysin (B) and ATPase-α (C): The signal of each band was normalized to the reference signal from the same blot. Data are mean ± SD, N = 3 independent biological samples for each condition with 3 technical repeats per sample.</p

HNE-damage to synaptosomal protein from vulnerable regions of the KO does not exceed damage in controls.

<p>ROS damage to synaptosomes was assessed by SDS PAGE Western blots probed for HNE reaction products. A blot (A) with 12 independent brainstem samples is shown as an example. (See supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148219#pone.0148219.s003" target="_blank">S3 Fig</a>. for the full set.) The positions of the 3 most immunoreactive bands are labeled (HNE) showing an unidentified band at 53kD band as the predominant reaction product. Non-specific bands (non-spec.) were independent of the primary antibody. Densitometry results (B) for all brain regions: For each sample the 53kD signal was normalized to the reference sample (“Ref”), an identical aliquot of which was run on each blot. The number of biological replicates is stated at the bottom of each bar. * denotes significant (α = 5%) differences after Holm-Bonferroni correction for multiple comparisons.</p