N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

NAT8L (N-acetyltransferase 8-like) accelerates lipid turnover and increases energy expenditure in brown adipocytes.

NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.

Large Timescale Interrogation of Neuronal Function by Fiberless Optogenetics Using Lanthanide Micro-particles.

Optogenetics requires implantation of light-delivering optical fibers, as current light-sensitive opsins are activated by visible light, which cannot effectively penetrate biological tissues. Insertion of optical fibers and subsequent photostimulation inherently damages brain tissue, and fiber tethering can restrict animal behavior. To overcome these technical limitations, we developed minimally invasive "fiberless" optogenetics using lanthanide micro-particles (LMPs), which emit up-conversion luminescence in the visible spectrum in response to irradiation with tissue-penetrating near-infrared light. Depolarizing (C1V1) and hyperpolarizing (ACR1) opsins were strongly activated by up-conversion luminescence from green-emitting LMPs both in vitro and in vivo. Using this technique, we successfully manipulated locomotive behavior of mice by activating and inhibiting neurons in the dorsal striatum, at a depth of 2 mm from the brain surface. LMPs were retained and remained functional for >8 weeks at the injection site. Fiberless optogenetics offers opportunities to control neuronal function over longer time frames using freely behaving animals.

Vulnerability to depressive behavior induced by overexpression of striatal Shati/Nat8l via the serotonergic neuronal pathway in mice.

The number of patients with depressive disorders is increasing. However, the mechanism of depression onsets has not been completely revealed. We previously identified Shati/Nat8l, an N-acetyltransferase, in the brain using an animal model of psychosis. In this study, we revealed the involvement of Shati/Nat8l in the vulnerability to major depression. Shati/Nat8l mRNA was increased only in the striatum of mice, which were exposed to chronic social defeat stress. Shati/Nat8l-overexpressed mice showed impairment in social interaction and sucrose preference after the subthreshold social defeat (microdefeat) stress. These depression-like behaviors were restored by fluvoxamine and LY341495 injection prior to these tests. Furthermore, the intracerebral administration of only fluvoxamine, but not of LY341495, to the dorsal striatum and direct infusion of LY341495 to the dorsal raphe also rescued. Taken together, Shati/Nat8l in the striatum has an important role in the vulnerability to depression onsets by regulating the origin of serotonergic neuronal system via GABAergic projection neuron in the dorsal raphe from the dorsal striatum.

GABA neurons in the ventral tegmental area regulate non-rapid eye movement sleep in mice.

Sleep/wakefulness cycle is regulated by coordinated interactions between sleep- and wakefulness-regulating neural circuitry. However, the detailed mechanism is far from understood. Here, we found that glutamic acid decarboxylase 67-positive GABAergic neurons in the ventral tegmental area (VTAGad67+) are a key regulator of non-rapid eye movement (NREM) sleep in mice. VTAGad67+ project to multiple brain areas implicated in sleep/wakefulness regulation such as the lateral hypothalamus (LH). Chemogenetic activation of VTAGad67+ promoted NREM sleep with higher delta power whereas optogenetic inhibition of these induced prompt arousal from NREM sleep, even under highly somnolescent conditions, but not from REM sleep. VTAGad67+ showed the highest activity in NREM sleep and the lowest activity in REM sleep. Moreover, VTAGad67+ directly innervated and inhibited wake-promoting orexin/hypocretin neurons by releasing GABA. As such, optogenetic activation of VTAGad67+ terminals in the LH promoted NREM sleep. Taken together, we revealed that VTAGad67+ play an important role in the regulation of NREM sleep.

REM sleep-active MCH neurons are involved in forgetting hippocampus-dependent memories.

The neural mechanisms underlying memory regulation during sleep are not yet fully understood. We found that melanin concentrating hormone-producing neurons (MCH neurons) in the hypothalamus actively contribute to forgetting in rapid eye movement (REM) sleep. Hypothalamic MCH neurons densely innervated the dorsal hippocampus. Activation or inhibition of MCH neurons impaired or improved hippocampus-dependent memory, respectively. Activation of MCH nerve terminals in vitro reduced firing of hippocampal pyramidal neurons by increasing inhibitory inputs. Wake- and REM sleep-active MCH neurons were distinct populations that were randomly distributed in the hypothalamus. REM sleep state-dependent inhibition of MCH neurons impaired hippocampus-dependent memory without affecting sleep architecture or quality. REM sleep-active MCH neurons in the hypothalamus are thus involved in active forgetting in the hippocampus.

Methamphetamine induces Shati/Nat8L expression in the mouse nucleus accumbens via CREB- and dopamine D1 receptor-dependent mechanism.

Shati/Nat8L significantly increased in the nucleus accumbens (NAc) of mice after repeated methamphetamine (METH) treatment. We reported that Shati/Nat8L overexpression in mouse NAc attenuated METH-induced hyperlocomotion, locomotor sensitization, and conditioned place preference. We recently found that Shati/Nat8L overexpression in NAc regulates the dopaminergic neuronal system via the activation of group II mGluRs by elevated N-acetylaspartylglutamate following N-acetylaspartate increase due to the overexpression. These findings suggest that Shati/Nat8L suppresses METH-induced responses. However, the mechanism by which METH increases the Shati/Nat8L mRNA expression in NAc is unclear. To investigate the regulatory mechanism of Shati/Nat8L mRNA expression, we performed a mouse Shati/Nat8L luciferase assay using PC12 cells. Next, we investigated the response of METH to Shati/Nat8L expression and CREB activity using mouse brain slices of NAc, METH administration to mice, and western blotting for CREB activity of specific dopamine receptor signals in vivo and ex vivo. We found that METH activates CREB binding to the Shati/Nat8L promoter to induce the Shati/Nat8L mRNA expression. Furthermore, the dopamine D1 receptor antagonist SCH23390, but not the dopamine D2 receptor antagonist sulpiride, inhibited the upregulation of Shati/Nat8L and CREB activities in the mouse NAc slices. Thus, the administration of the dopamine D1 receptor agonist SKF38393 increased the Shati/Nat8L mRNA expression in mouse NAc. These results showed that the Shati/Nat8L mRNA was increased by METH-induced CREB pathway via dopamine D1 receptor signaling in mouse NAc. These findings may contribute to development of a clinical tool for METH addiction.