Shati/Nat8l and N-acetylaspartate (NAA) Have Important Roles in Regulating Nicotinic Acetylcholine Receptors in Neuronal and Psychiatric Diseases in Animal Models and Humans
Shati/Nat8l was originally isolated as a methamphetamine-related-molecule from the nucleus accumbens of mice. Since then, Shati/Nat8l has been characterized as an N-acetyltransferase-8-like protein (Nat8l) that catalyzes N-acetylaspartate (NAA) synthesis from aspartate and acetyl-coenzyme A. It has been shown that elevated NAA levels detected by proton magnetic resonance spectroscopy (1H-MRS) brain imaging indicates increased neuronal activity. Our group produced Shati/Nat8l knock out mice (Shati/Nat8l KO mice), which exhibit hyper locomotion, anxiety behaviors, and social dysfunction. These mice have a high sensitivity to methamphetamine, as evidenced by their results in assessments of locomotor activity and conditioned place preference, as well as their elevated dopamine levels. We used an adeno-associated virus (AAV) vector containing Shati/Nat8l (AAV-Shati/Nat8l) to overexpress the protein in different brain regions such as the striatum and the nucleus accumbens, in order to investigate their involvement in methamphetamine-induced behavioral and pharmacological changes. We showed that overexpression of accumbal Shati/Nat8l attenuates methamphetamine-induced behaviors.
Recent clinical studies have revealed further novel roles of Shati/Nat8l in psychiatric and neuronal diseases. We are just beginning to appreciate the various actions of this intriguing, recently discovered molecule in the central nervous system.
KeywordsShati/Nat8l Methamphetamine Addiction Depression Alzheimer’s disease ATP
Furthermore, treatment of a neuroblastoma-derived cell line with a physiological level of NAA resulted in apoptosis of cancerous cells and enhanced neuronal differentiation (Mazzoccoli et al. 2016). NAA is of unique clinical significance and hence is exploited in MRS. Treatment of an SH-SY5Y neuroblastoma-derived cell line with sub-cytotoxic physiological concentrations of NAA has been shown to inhibits cell growth (Mazzoccoli et al. 2016). This effect is partly due to enhanced apoptosis, which is indicated by a decrease in the anti-apoptotic factors survivin and Bcl-xL, and partly due to the arrest of the cell-cycle progression, linked to enhanced expression of the cyclin-inhibitors p53, p21Cip1/Waf1, and p27Kip1 (Mazzoccoli et al. 2016).NAA-pre-treated SH-SY5Y cells were more sensitive to the cytotoxic effect of the chemotherapeutic drugs cisplatin and 5-fluorouracil (Mazzoccoli et al. 2016).
In this review, we will introduce the various functions of Shati/Nat8l and NAA in psychiatric behaviors, especially addictive behaviors, and summarize their roles in neuronal and psychiatric diseases.
6.2 Shati/Nat8l and Drug Reward
6.2.1 Function of Accumbal Shati/Nat8l in Nicotinic Effects
We previously reported that overexpression of Shati/Nat8l in the nucleus accumbens (NAc) of mice depressed the pharmacological effects of methamphetamine, especially addiction-related behaviors, hyperactivity, and place preference. In an in vivo microdialysis experiment, the extracellular dopamine (DA) level was increased by 200–300% of the base line by peripheral methamphetamine injection. In the NAc of these mice, NAA and NAAG levels were significantly reduced (Miyamoto et al. 2014). The mGluR3 antagonist, LY341495, cancelled the Shati-Nat8l-indeuced reduction of hyperlocomotion and conditioned place preference after methamphetamine treatment. Furthermore, LY341495 also cancelled the Shati/Nat8l-associated increase in extracellular DA levels in the NAc of methamphetamine-treated mice. These results indicate that the overexpression of Shati/Nat8l in the NAc suppresses the increase in dopamine release caused by methamphetamine via mGluR3 (Miyamoto et al. 2014).
Taken together, our results show that Shati/Nat8l in the NAc has a protective effect against the deleterious physiological changes associated with nicotine or methamphetamine administration.
6.2.2 Striatal Shati/Nat8l and the Reward System
6.3 Shati/Nat8l in Learning and Memory
6.3.1 Hippocampal Shati/Nat8l in Learning and Memory
6.3.2 Function of Accumbal Shati/Nat8l on Learning Memory and Emotional Behaviors
We have shown that nicotine reversed scopolamine-induced impairment in the passive avoidance task in rats through its action on the dopaminergic neuronal system (Nitta et al. 1994). The study did not mention which brain areas are important for the nicotine-potentiated learning ability. If altered DA levels triggered by Shati/Nat8l overexpression had any effect on cognitive function, Shati/Nat8l-Tg mice would show potentiated cognitive function over normal mice. However, their cognitive functions were not changed in Y maze or novel object tests (Figs. 6.8 and 6.9).
6.3.3 Function of Shati/Nat8l in Axon Outgrowth
In vitro studies were performed to evaluate the function of Shati/Nat8l in neuronal cells, since Shati/Nat8l mRNA signals were observed in all brain regions of mice (Sumi et al. 2015). Especially strong signals were found in cortical pyramidal cells, dentate granule cells, hippocampal pyramidal cells, and cerebellar granule cells. Neuronal cells of all brain regions showed positive Shati/Nat8l mRNA signals. Hippocampal neurons were selected for their easy evaluation and high cell density. Shati/Nat8l mRNA positive cells colocalized with NeuN (a marker for neurons)- positive cells but not with GFAP (a marker for astrocytes)- or Iba1 (a marker for microglia)-positive cells. These results show that Shati/Nat8l mRNA is only expressed in neuronal cells of the mouse hippocampus. An AAV vector containing Shati/Nat8l was transfected into primary cultured mouse neurons. Overexpression of Shati/Nat8l in primary cultured neurons induced axonal growth but not dendrite elongation. Treatment with a selective group II mGluR antagonist did not eliminate Shati/Nat8l-induced axon outgrowth, and NAAG itself did not induce axon outgrowth. Overexpression of Shati/Nat8l also increased the ATP content in the cultured hippocampal neurons. These results suggest that neuronal Shati/Nat8l induces axon outgrowth via ATP synthesis independently of the mGluR3 signaling pathway. Shati/Nat8l is associated with microtubule structure when overexpressed in COS7 cells and primary mouse cultured neurons (Toriumi et al. 2013). On the other hand, it was also reported that Shati/Nat8l co-localizes with a mitochondrial marker in SH-SH5Y cells (Ariyannur et al. 2010), and that Shati/Nat8l is localized in the endoplasmic reticulum (Wiame et al. 2009). Shati/Nat8l may have novel functions in neuronal cells. NAA is produced in the mitochondria because it is associated with the tricarboxylic acid cycle (TCA) related to cell metabolism (Madhavarao et al. 2003). The levels of NAA and ATP were increased in primary cultured neurons overexpressed with Shati/Nat8l. The TCA cycle produces ATP molecules at the highest rate in terms of cell metabolism, and ATP in the growth cone is known to promote neurite elongation in cultured neurons (Höpker et al. 1996). Neuronal dendrite length in Shati/Nat8l KO mice was significantly shorter than in wildtype mice (Berent-Spillson et al. 2004). Shati/Nat8l appears to play a major role in ATP-induced neurite elongation. Shati/Nat8l is an indicator of the stimulation of mGluR3. However, neither NAAG nor LY341495, the endogenous agonist of mGluR3 and an antagonist of mGluR3, respectively, affected axon outgrowth. Shati/Nat8l is thus associated with neurite elongation and the ATP synthesis pathway during NAA synthesis.
6.4 Shati/Nat8l and Psychiatric Disease
6.4.1 Patients with Depression and NAA
NAA is used as a biomarker of depression in specific regions of the human brain as it is an indicator of neuronal activity in 1H–MRS. NAA is significantly decreased in the anterior cingulate gyrus of patients with depression. Brain-derived neurotrophic factor (BDNF) has been reported to be one of the biomarkers for patients with depressive (Rogóż et al. 2017; Zhao et al. 2016). Anti-depressant drugs rescue the reduction in NAA, in addition to BDNF. Interestingly, BDNF mRNA is increased in the prefrontal cortex, the NAc and the hippocampus in the brains of Shati/Nat8l KO mice (Furukawa-Hibi et al. 2012). Therefore, the changes in Shati/Nat8l and BDNF are not parallel, but independent. Although detailed mechanisms for the roles of Shati/Nat8l and/or BDNF in depression have not been clarified, they may nevertheless serve as reliable clinical markers.
6.4.2 Shati/Nat8l and Depressive Behaviors in Mice
Miyamoto et al. (2017) also demonstrated that striatal Shati/Nat8l induces depressive behaviors. We produced mice overexpressing Shati/Nat8l in the striatum (Str-Shati/Nat8l mice) using an AAV vector, as described earlier. The Str-Shati/Nat8l mice showed depression-like behaviors in forced swimming and tail suspension tests (Miyamoto et al. 2017) as measured by prolonged immobility, indicating that Shati/Nat8l is an inducer of depression. These dysfunctions are cancelled by the anti-depressant drug, fluvoxamine, and the mGluR3 antagonist, LY341495. Shati/Nat8l levels may be a factor in the vulnerability to stress. In the psychiatric field, mental diseases are often caused by two factors, genetic background and circumstance, and striatal Shati/Nat8l might thus represent one of the genetic factors.
6.4.3 Shati/Nat8l and Postpartum Depression
Postpartum depression is observed in about 13% of postpartum women, and is defined as a depressive disorder leading to a substantial impairment of daily life. It also has large impacts on the patients’ families, including the promotion of depressive tendencies in the husband, the abuse or neglect of the child and/or delayed cognitive development or increased psychopathological issues in the child (Stumbo et al. 2015).
We also carried out a clinical study among pregnant women. Serum concentrations of Shati/Nat8l were measured in pregnant womean during late pregnancy and at 5 days and 1 month after delivery, respectively. The women were divided into two groups, a non-depressive and a depressive group, based on their score on the Edinburgh Postnatal Depression Scale (EPDS). Serum concentrations of Shati/Nat8l were higher in the depressive group than in the non-depressive group at the time point before delivery (Nitta et al. unpublished data). Serum concentrations of Shati/Nat8l at the late pregnancy stage could be one predictive biomarker for postnatal depression, and the appropriate preventive and early interventions might thus be undertaken for pregnant and postpartum women. We also measured serum concentrations of Shati/Nat8l in these women after delivery, but observed no significant differences.
Shati/Nat8l and NAA have various functions in the central nervous system. In addition to the neuronal system, Shati/Nat8l is also highly expressed in the adipose tissue, and NAA pathway could similarly serve as an acetyl-CoA metabolizing mechanism in adipocytes (Pessentheiner et al. 2013). An increase in lipolysis followed by an activation of β-oxidation can restore acetyl-CoA back to the mitochondria. Lipid turnover can raise the oxidative potential of the brown fat cells and thereby boost the brown adipogenic phenotype. However, the physiological stimuli contributing to the regulation of the NAA pathway are unknown (Pessentheiner et al. 2013). These results suggest the possibility that Shati/Nat8l also plays a role in lifestyle diseases such as diabetes.
Because of their widespread occurrence in the human body, Shati/Nat8l and NAA have the potential to be used as treatment tools for a variety of diseases in the near future.
Conflicts of Interest
The authors have declared that no competing interests exist.
This study was supported by a Smoking Research Foundation Grant for Biomedical Research and Foundation, the Program for Next Generation World-Leading Researchers [NEXT Program LS047], a grant-in-aid for Scientific Research (KAKENHI) (B) [JSPS KAKENHI Grant Number, JP15H04662], a Challenging Exploratory Research grant [JSPS KAKENHI grant number, JP15K15050; 17 K19801] from the Japan Society for the Promotion of Science, a Research on Regulatory Science of Pharmaceuticals and Medical Devices grant from the Japan Agency for Medical Research and Development (AMED) [16mk0101076h0001], and the Kobayashi International Foundation.
We thank Naomi Takino, Hitomi Miyauchi, and Keiko Ayabe for technical assistance in producing the AAV-Shati/Nat8l vectors.
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