Circadian Rheb oscillation alters the dynamics of hepatic mTORC1 activity and mitochondrial morphology.

The morphological structure and metabolic activity of mitochondria are coordinately regulated by circadian mechanisms. However, the mechanistic interplay between circadian mechanisms and mitochondrial architecture remains poorly understood. Here, we demonstrate circadian rhythmicity of Rheb protein in liver, in line with that of Per2. Using genetic mouse models, we show that Rheb, a small GTPase that binds mTOR, is critical for circadian oscillation of mTORC1 activity in liver. Disruption of Rheb oscillation in hepatocytes by persistent expression of Rheb transgene interrupted mTORC1 oscillation. We further show that Rheb-regulated mTORC1 altered mitochondrial fission factor DRP1 in liver, leading to altered mitochondrial dynamics. Our results suggest that Rheb/mTORC1 regulated DRP1 oscillation involves ubiquitin-mediated proteolysis. This study identifies Rheb as a nodal point that couples circadian clock and mitochondrial architecture for optimal mitochondrial metabolism.

Rheb (Ras Homolog Enriched in Brain 1) Deficiency in Mature Macrophages Prevents Atherosclerosis by Repressing Macrophage Proliferation, Inflammation, and Lipid Uptake.

OBJECTIVE: Macrophage foam cell formation is an important process in atherosclerotic plaque development. The small GTPase Rheb (Ras homolog enriched in brain 1) regulates endocytic trafficking that is critical for foam cell formation. However, it is unclear whether and how macrophage Rheb regulates atherogenesis, which are the focuses of the current study. Approach and Results: Immunofluorescence study confirmed the colocalization of Rheb in F4/80 and Mac-2 (galectin-3)-labeled lesional macrophages. Western blot and fluorescence-activated cell sorting analysis showed that Rheb expression was significantly increased in atherosclerotic lesions of atherosclerosis-prone (apoE-/- [apolipoprotein E deficient]) mice fed with Western diet. Increased Rheb expression was also observed in oxidized LDL (low-density lipoprotein)-treated macrophages. To investigate the in vivo role of macrophage Rheb, we established mature RhebmKO (macrophage-specific Rheb knockout) mice by crossing the Rheb floxed mice with F4/80-cre mice. Macrophage-specific knockout of Rheb in mice reduced Western diet-induced atherosclerotic lesion by 32%, accompanied with a decrease in macrophage content in plaque. Mechanistically, loss of Rheb in macrophages repressed oxidized LDL-induced lipid uptake, inflammation, and macrophage proliferation. On the contrary, lentivirus-mediated overexpression of Rheb in macrophages increased oxidized LDL-induced lipid uptake and inflammation, and the stimulatory effect of Rheb was suppressed by the mTOR (mammalian target of rapamycin) inhibitor rapamycin or the PKA (protein kinase A) activator forskolin. CONCLUSIONS: Macrophage Rheb plays important role in Western diet-induced atherosclerosis by promoting macrophage proliferation, inflammation, and lipid uptake. Inhibition of expression and function of Rheb in macrophages is beneficial to prevent diet-induced atherosclerosis.

Effects of antiepileptic drugs in a new TSC/mTOR-dependent epilepsy mouse model.

OBJECTIVE: An epilepsy mouse model for Tuberous Sclerosis Complex (TSC) was developed and validated to investigate the mechanisms underlying epileptogenesis. Furthermore, the possible antiepileptogenic properties of commonly used antiepileptic drugs (AEDs) and new compounds were assessed. METHODS: Tsc1 deletion was induced in CAMK2A-expressing neurons of adult mice. The antiepileptogenic properties of commonly used AEDs and inhibitors of the mTOR pathways were assessed by EEG recordings and by molecular read outs. RESULTS: Mice developed epilepsy in a narrow time window (10 ± 2 days) upon Tsc1 gene deletion. Seizure frequency but not duration increased over time. Seizures were lethal within 18 days, were unpredictable, and did not correlate to seizure onset, length or frequency, reminiscent of sudden unexpected death in epilepsy (SUDEP). Tsc1 gene deletion resulted in a strong activation of the mTORC1 pathway, and both epileptogenesis and lethality could be entirely prevented by RHEB1 gene deletion or rapamycin treatment. However, other inhibitors of the mTOR pathway such as AZD8055 and PF4708671 were ineffective. Except for ketogenic diet, none of commonly used AEDs showed an effect on mTORC1 activity. Vigabatrin and ketogenic diet treatment were able to significantly delay seizure onset. In contrast, survival was shortened by lamotrigine. INTERPRETATION: This novel Tsc1 mouse model is highly suitable to assess the efficacy of antiepileptic and -epileptogenic drugs to treat mTORC1-dependent epilepsy. Additionally, it allows us to study the mechanisms underlying mTORC1-mediated epileptogenesis and SUDEP. We found that early treatment with vigabatrin was not able to prevent epilepsy, but significantly delayed seizure onset.

RHEB1 insufficiency in aged male mice is associated with stress-induced seizures.

The mechanistic target of rapamycin (mTOR), a protein kinase, is a central regulator of mammalian metabolism and physiology. Protein mTOR complex 1 (mTORC1) functions as a major sensor for the nutrient, energy, and redox state of a cell and is activated by ras homolog enriched in brain (RHEB1), a GTP-binding protein. Increased activation of mTORC1 pathway has been associated with developmental abnormalities, certain form of epilepsy (tuberous sclerosis), and cancer. Clinically, those mTOR-related disorders are treated with the mTOR inhibitor rapamycin and its rapalogs. Because the effects of chronic interference with mTOR signaling in the aged brain are yet unknown, we used a genetic strategy to interfere with mTORC1 signaling selectively by introducing mutations of Rheb1 into the mouse. We created conventional knockout (Rheb1 +/- ) and gene trap (Rheb1 Δ/+ ) mutant mouse lines. Rheb1-insufficient mice with different combinations of mutant alleles were monitored over a time span of 2 years. The mice did not show any behavioral/neurological changes during the first 18 months of age. However, after aging (> 18 months of age), both the Rheb1 +/- and Rheb1 Δ /- hybrid males developed rare stress-induced seizures, whereas Rheb1 +/- and Rheb1 Δ /- females and Rheb1 Δ/+ and Rheb1 Δ/Δ mice of both genders did not show any abnormality. Our findings suggest that chronic intervention with mTORC1 signaling in the aged brain might be associated with major adverse events.