An L-Glutamine Transporter Isoform for Neurogenesis Facilitated by L-Theanine

Neurochem Res DOI 10.1007/s11064-017-2317-6

REVIEW PAPER

An l-Glutamine Transporter Isoform for Neurogenesis Facilitated by l-Theanine

Yukio Yoneda1

Received: 7 May 2017 / Revised: 25 May 2017 / Accepted: 29 May 2017 © Springer Science+Business Media New York 2017

Abstract l-Theanine (=γ-glutamylethylamide) is an on the development of dietary supplements and nutraceuti- amino acid ingredient in green tea with a structural anal- cals enriched of l-theanine for the prophylaxis of a variety ogy to l-glutamine (l-GLN) rather than l-glutamic acid of untoward impairments and malfunctions seen in patients (l-GLU), with regards to the absence of a free carboxylic with different neurodegenerative and/or neuropsychiatric acid moiety from the gamma carbon position. l-theanine disorders. 3 markedly inhibits [ H]l-GLN uptake without affecting 3 [ H]l-GLU uptake in cultured neurons and astroglia. In Keywords l-Theanine · l-Glutamine · Neural stem cells · neural progenitor cells with sustained exposure to l-thean- bHLH genes · mTOR · Neurogenesis ine, upregulation of the l-GLN transporter isoform Slc38a1 expression and promotion of both proliferation and neu- Abbreviations ronal commitment are seen along with marked acceleration ADHD Attention deficit hyperactivity disorder of the phosphorylation of mammalian target of rapamycin AP1 Activator protein-1 (mTOR) and relevant downstream proteins. Stable overex- bHLH Basic helix-loop-helix pression of Slc38a1 leads to promotion of cellular growth BrdU 5-bromo-2′-deoxyuridine with facilitated neuronal commitment in pluripotent embry- CREB Cyclic AMP responsive element binding protein onic carcinoma P19 cells. In P19 cells stably overexpress- DG Dentate gyrus ing Slc38a1, marked phosphorylation is seen with mTOR EAA Essential amino acids and downstream proteins in a fashion insensitive to the ER Endoplasmic reticulum additional stimulation by l-theanine. The green tea amino GABA γ-aminobutyric acid acid l-theanine could thus elicit pharmacological actions GFAP Glial fibrillary acidic protein to up-regulate Slc38a1 expression for activation of the GFP Green fluorescent protein mTOR signaling pathway required for cell growth together GLN Glutamine with accelerated neurogenesis after sustained exposure in GlnT GLN transporter undifferentiated neural progenitor cells. In this review, I GLU Glutamic acid summarize a novel pharmacological property of the green MAP2 Microtubule-associated protein-2 tea amino acid l-theanine for embryonic and adult neuro- MeAIB N-methylaminoisobutyric acid genesis with a focus on the endogenous amino acid analog mTOR Mammalian target of rapamycin l-GLN. A possible translational strategy is also discussed MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide NSCs Neural stem cells * Yukio Yoneda Slc38a1 Solute carrier 38a1 [email protected]‑u.ac.jp XBP1 X-box binding protein-1

1 Section of Prophylactic Pharmacology, Kanazawa University Venture Business Laboratory 402, Kanazawa, Ishikawa 920– 1192, Japan

Vol.:(0123456789)1 3 Neurochem Res

Introduction excitation by caffeine of brain excitability as revealed by electroencephalography in rats with an intravenous injec- l-theanine (=γ-glutamylethylamide) is an amino acid tion of l-theanine [8]. l-theanine efficiently suppressed ele- ingredient of green tea with a chemical structure analogous vations of systemic blood pressure and brain 5-hydroxyin- to l-glutamine (l-GLN) rather than l-glutamic acid (l- doles levels in spontaneous hypertensive rats [9]. A prior GLU) [1]. l-GLU has been thought to play a dual role as an intracerebroventricular injection of l-theanine protected excitotoxin [2] and as an excitatory neurotransmitter in the the hippocampal CA1 pyramidal neurons against delayed brain, while a careful structural evaluation clearly reveals a neuronal cell death in gerbils with bilateral forebrain global high similarity of l-theanine to l-GLN, which is the princi- ischemia [10], whereas l-theanine was a weak displacer of pal precursor of l-GLU and γ-aminobutyric acid (GABA) ligand binding to three different neurotoxic receptor sub- in the brain [3]. As seen with the structure of l-GLN, a free types, compared to l-GLU, in rat cortical synaptic mem- carboxylic acid moiety is absent from the gamma carbon branes [11]. These included glutamate receptor subtypes position in l-theanine (Fig. 1). Although l-theanine has sensitive to NMDA, AMPA and KA with high neurotoxic- been thought to modulate both neurotransmission and neu- ity [12]. rotoxicity mediated by l-GLU in the literature [4, 5], the In healthy human subjects, l-theanine is shown to elicit aforementioned structural similarity gives rise to an idea protective effects on different psychological and physi- that l-GLN would be at least in part responsible for par- ological stress responses [13]. In human adults with physi- ticular pharmacological properties of l-theanine, besides l- cal and psychological stress tasks, for instance, oral intake GLU, in the brain. of l-theanine was more effective in ameliorating increases in both anxiety and blood pressure than the other green tea ingredient caffeine [14]. In patients with major depressive The Green Tea Amino Acid l‑theanine disorders, chronic administration of l-theanine for 8 weeks induced several beneficial effects on depressive symptoms, High-quality green tea, such as “Gyokuro” and “Matcha”, anxiety, sleep disturbance and cognitive impairments [15]. contains l-theanine (=γ-glutamylethylamide) by more than In boys diagnosed as attention deficit hyperactivity disor- 2 up to 3% [6]. This amount is considerably higher than the der (ADHD), daily oral intake of l-theanine for 6 weeks content in other tea beverages, such as black tea and oolong improved some aspects of sleep quality [16]. Similar alle- tea, from the identical tea plant “Chanoki” (Camellia sin- viation is seen in ADHD-related sleep disorders with ensis). The amount of daily l-theanine intake is estimated regards to sleep efficiency rather than total sleep time [17]. to be about 20 mg after the ingestion of 1 g of powdered Accumulating evidence is available for the promotion by green tea (Matcha) used for the traditional tea ceremony in l-theanine of good quality of sleep through anxiolysis and Japan. In contrast to acidic l-GLU, l-theanine easily gained relaxation without remarkable adverse side effects in the access to the brain through the blood brain barrier in con- literature [18]. scious rats [7]. Indeed, alleviation was seen in excessive

The Endogenous Amino Acid l‑GLN

l‑GLN Transporters

It should be emphasized that l-GLN was actively incorporated into intracellular spaces in rat brain slices [19]. Sev- eral independent lines of evidence indicate the involvement of different membrane transporters in active transmembrane migration of l-GLN. At least three discrete sodium- dependent transport systems are believed to mediate l-GLN transport across plasma membranes in the brain as summa- rized in Table 1; (1) the system A [20–22]; (2) the system ASC [23, 24] and (3) the system N [25, 26]. In addition, l- Fig. 1 Comparison of chemical structures of l-theanine, l-GLN, GLN was taken up into the cytoplasm through the sodium- l-GLU and GABA. l-theanine is more analogous to l-GLN than l- independent system L composed of heteromeric assemblies GLU in terms of the absence of a free carboxylic acid moiety from of different subunits [27–29]. Amongst these four differ- the gamma carbon position. In the brain, l-GLN is transformed into l l-GLU by glutaminase, along with further conversion of l-GLU into ent transport systems for carrying -GLN across plasma GABA by l-GLU decarboxylase membranes, the l-GLN transporter (GlnT; =ATA1/SAT1/

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Table 1 Classification of GLN System Gene Protein Substrate transporters System A Slc38a1 SNAT1 (=GlnT) Gln; Ala; Cys; His; Ser Slc38a2 SNAT2 Gln; Ala; Cys; Gly; His; Met; Pro; Ser Slc38a4 SNAT4 Ala; Asn; Cys; Gly; Ser; Thr System ASC Slc1a5 ASCT2 Gln; Ala; Ser; Cys System N Slc38a3 SNAT3 Gln; His Slc38a5 SNAT5 Gln; Asn; Ser; His System L Slc7a5 LAT1 Leu; Ile; Val; Phe; Tyr; Trp; Met; His Slc7a8 LAT2 Most neutral amino acids

SNAT1) classified as a member the system A family is sup- membranes from rat brain [1]. However, no marked binding 3 posed to be exclusively expressed by neurons with high activity for [ H]l-theanine was detected in synaptic mem- affinity forl -GLN in the brain [30–32]. branes in a manner independent of incubation time and temperature. In contrast to the null receptor binding activity 3 l‑GLU/l‑GLN Cycle in membranes, [ H]l-theanine was highly accumulated in a temperature-dependent manner in rat brain synaptosomes. 3 The view that l-GLN exported from astrocytes to extra- Saturation isotherms revealed that [ H]l-theanine accumu- cellular spaces is taken up through the aforementioned lation consisted of at least two different components with l-GLN transporter GlnT expressed by neurons to fuel the Km values of 42.3 µM and 1.88 mM, and Vmax values of l-GLU/l-GLN cycle required for the neurotransmitter 3.92 and 104.2 nmol/h/mg protein, respectively [1]. The 3 pool of l-GLU at glutamatergic nerve terminals is widely fact that l-theanine failed to affect [ H]l-GLU accumula- accepted. In the central nervous system, l-GLN is thought tion in synaptosomes gives support for the idea that l-thea- to be the principal substrate for GlnT to provide a precur- nine is incorporated into rat brain synaptosomes through sor for the synthesis of the neurotransmitters l-GLU and a mechanism not related to any transporter isoforms for GABA [3]. In neurons, l-GLN is hydrolyzed to l-GLU by l-GLU. 3 the catalytic action of phosphate-dependent glutaminase Indeed, [ H]l-theanine accumulation was selectively [3] for the condensation into synaptic vesicles by vesicular inhibited by the addition of the other structural analog, l- glutamate transporters expressed at vesicular surfaces for GLN, in a concentration-dependent manner at concentra- subsequent exocytotic release into synaptic clefts. Extracel- tion ranges of 10 nM–1 mM, which occurred to a greater lular l-GLU is then removed from synaptic clefts by excita- extent than the inhibition by l-theanine itself [1]. In syn- 3 tory amino acid transporters expressed by adjacent cells aptosomes, [ H]l-GLN was also accumulated in a tem- including neurons and astrocytes. The latter active trans- perature-dependent and saturable manner. The addition of 3 port is highly responsible for preventing the accumulation l-theanine markedly inhibited [ H]l-GLN accumulation in of extracellular l-GLU to a neurotoxic level, in addition to a concentration-dependent fashion at concentrations of 10 terminating glutamatergic neurotransmission at synapses. nM–1 mM. Similar pharmacological profiles were invari- In astrocytes, l-GLU incorporated is converted to l-GLN ably seen with other endogenous amino acids in inhibiting 3 3 by l-GLN synthetase, followed by efflux of l-GLN into the accumulation of both [ H]l-theanine and [ H]l-GLN 3 extracellular spaces from astrocytes and subsequent reentry [1]. Accordingly, [ H]l-theanine could be incorporated 3 mediated by GlnT into neurons to fuel the above-mentioned through a mechanism relevant to [ H] l-GLN accumulation l-GLU/l-GLN cycle at glutamatergic nerve terminals [3, in rat brain synaptosomes. 29, 33]. However, l-theanine was not a good substrate for The mutual inhibition profile supports the assumption phosphate-dependent glutaminase enriched in the brain, that extracellular l-theanine could at least in part modu- but metabolized by phosphate-independent glutaminase late extracellular and intracellular levels of l-GLN under responsible for the degradation of l-GLN in the kidney the delicate control by GlnT expressed in cells adjacent to [34]. glutamatergic synapses in a particular pathological situation such as brain ischemia. A higher level of l-GLN was Common Transporters for l‑theanine and l‑GLN found in the cerebrospinal fluid of patients with Alzhei- mer’s disease than in normal subjects [35]. Taken together, To confirm the accessibility of l-theanine for particular l-theanine would suppress glutamatergic neurotransmis- membrane receptors other than neurotoxic glutamate recep- sion through a mechanism related to the inhibition of l- 3 tors, [ H]l-theanine was incubated with crude synaptic GLN incorporation across plasma membranes into neurons

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3 3 toward subsequent neuroprotection against delayed neu- Both [ H]l-theanine and [H]l-GLN were similarly ronal cell death mediated by extracellular l-GLU after accumulated in synaptosomes of telencephalic structures ischemia. In fact, we have demonstrated that l-theanine sig- including neocortex, hippocampus and corpus striatum nificantly decreased the extracellular levels of endogenous in a sodium-dependent manner, with progressively less l-GLU in cultured rat cortical neurons [1]. The possibility potent activities in midbrain, hypothalamus, medulla- 3 that l-theanine has high affinities for particular isoforms pons and cerebellum [1]. Similarly, both [ H]l-theanine 3 other than GlnT of membrane transporters capable of trans- and [ H]l-GLN were highly accumulated with a plateau porting l-GLN, however, remains to be elucidated in future within 60 min in primary cultured neurons and astrocytes studies. [1]. In both cell cultures, l-theanine markedly inhibited 3 [ H]l-GLN accumulation in a concentration-dependent manner. Sustained exposure to l-theanine led to a slight but The l‑GLN Transporter Slc38a1 (=GlnT) statistically significant decrease in the extracellular level of l-GLU in cultured neurons [1]. l-theanine could thus sup- The l-GLN transporter GlnT is the first member of the sys- press the spontaneous and/or exocytotic release of l-GLU tem A neutral amino acid transporter superfamily identi- supplied by the l-GLU/l-GLN cycle through a mechanism fied at the molecular level as solute carrier 38a1 (Slc38a1). associated with the inhibition of l-GLN incorporation in Of different transporters cloned to date, Slc38a1 has the glutamatergic neurons in a particular situation as described highest affinity forl -GLN with a sodium/amino acid above. The green tea amino acid l-theanine is thus sup- cotransport mechanism [36]. Another typical feature is the posed to be an inhibitor of Slc38a1 capable of transporting selective inhibition by the amino acid analog N-methylami- l-GLN across plasma membranes in neurons and astroglia. noisobutyric acid (MeAIB) of the l-GLN transport medi- The aforementioned low affinity and clear structural dis- ated by Slc38a1 [37, 38]. Constitutive expression was not crimination are not in favor of an idea for l-theanine to be found for Slc38a1 mRNA in primary cultured mouse neo- a modulator of the activity of particular ionotropic receptor cortical and cerebellar astrocytes, while marked expression subtypes for l-GLU in the brain as described previously [4, of Slc38a1 mRNA was seen in cultured neocortical neurons 5]. and cerebellar granular cells [33]. In these cultured astro- 3 cytes, [ H]l-GLN accumulation was insensitive to the inhibition by several system A substrates including MeAIB, Neural Stem Cells (NSCs) proline and glycine. In neurons, therefore, the system A transporter Slc38a1 seems to predominantly mediate trans- Mouse Embryonal Carcinoma Cells membrane transport of l-GLN. However, recent studies have shown constitutive expression of Slc38a1 mRNA in The fact that constitutive expression of Slc38a1 was simi- 3 cultured astrocytes prepared from rat whole brain together larly seen with activities to transport [ H]l-GLN in both 3 with [ H]l-GLN uptake and efflux activities insensitive to neurons and astroglia focused our attention on its possible MeAIB [39, 40]. Double labeling immunohistochemistry role in NSCs in embryonic, postnatal, developing and adult demonstrated the co-localization of immunoreactivities brains. Neural stem cells are defined as primitive progeni- for Slc38a1 and the glial marker protein, glial fibrillary tor cells endowed to proliferate for self-replication and to acidic protein (GFAP), in the cerebral cortex of adult rat differentiate into several progeny cell lineages including and human brains [41]. Constitutive expression of Slc38a1 neurons, astrocytes and oligodendrocytes [45–49]. Tak- 3 mRNA was seen along with MeAIB-sensitive [ H]l-GLN ing into consideration the fact that l-theanine is commonly 3 accumulation in cultured rat neocortical astrocytes [42], effective in inhibiting [ H]l-GLN incorporation in a con- whereas both transient upregulation [43] and stable overex- centration-dependent manner in cultured rat neocortical pression [44] of Slc38a1 invariably led to the exacerbation neurons and astrocytes, it is conceivable that the transporter of vulnerability to the oxidative cytotoxicity in cultured Slc38a1 would be constitutively and functionally expressed astrocytes. Transmembrane l-GLN transportation would be by NSCs capable of differentiating into neurons and astro- mediated by Slc38a1 belonging to the system A superfam- cytes. Indeed, both neurons and astrocytes were able to 3 3 ily, in addition to different transporters classified into the transport [ H]l-GLN and [ H]l-theanine with similar phar- system ASC, system N, and system L families, which were macological profiles [1]. all capable of transporting l-GLN across plasma mem- Mouse embryonal carcinoma P19 cells [50] are shown branes even in astrocytes. Astrocytes might play a role in to feature a phenotype similar to that of cells of the primi- mechanisms underlying regulation of the extracellular lev- tive ectoderm together with an ability to differentiate into els of l-GLN required for the l-GLU/l-GLN cycle in gluta- neural lineages by retinoic acid and into muscular cellular matergic neurons. lineages by dimethylsulfoxide, respectively [50, 51]. In the

1 3 Neurochem Res absence of differentiation inducers, P19 cells rapidly grow in undifferentiated P19 cells,l -theanine failed to further for clusters and express several early embryonic markers promote these elevated activities of proliferation, neuronal without limitation in culture. Culture with retinoic acid commitment and NeuroD1 gene expression in undifferenti- leads to the formation of round spheres composed of clus- ated P19 cells with stable overexpression of Slc38a1 [52]. tered proliferating cells with growing sizes proportional to These findings give support to the proposal that l-theanine the culture period in pluripotent carcinoma P19 cells under could promote both proliferation and neuronal commitment non-adherent conditions. Dispersion and subsequent cul- through a mechanism common to that underlying the pro- ture without retinoic acid led to differentiation into cells motion by stable Slc38a1 overexpression in undifferenti- immunoreactive for the neuronal marker microtubules- ated pluripotent P19 cells. associated protein-2 (MAP2), followed by appearance of cells immunoreactive for the astroglial marker GFAP under Upregulation of bHLH Transcription Factors adherent conditions. In undifferentiated P19 cells with sustained exposure to l-theanine, a marked increase was seen The drastic upregulation of both activator and repressor in their proliferation activity as determined by both the size types of bHLH genes argues in favor of an idea that par- of spheres composed of clustered proliferating cells and ticular bHLH transcription factors are responsible for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium the promotion of both proliferation for self-replication bromide (MTT) reducing activity [52]. Prior exposure to and neuronal commitment in stable Slc38a1 transfect- l-theanine, moreover, induced an increase in the number ants. The activator type bHLH factor NeuroD1 was abso- of cells immunoreactive for MAP2 with a concomitant lutely required for the survival and neuronal differentiation decrease in the number of cells immunoreactive for GFAP of neural stem cells during adult neurogenesis [54, 55], in P19 cells after spontaneous differentiation [52]. The pos- while NeuroD1 was identified as a gene highly relevant to sibility that l-theanine is able to promote both proliferation the terminal differentiation into neurons in postnatal and and subsequent neuronal differentiation with attenuated adult neurogenesis [56]. Overexpression of NeuroD1 led astroglial differentiation in undifferentiated NSCs is thus to rapid appearance of cells featuring morphological and conceivable. molecular properties of matured neurons in vivo, together with marked inhibition of terminal neuronal differentiation Stable Overexpression of Slc38a1 in Pluripotent P19 by shRNA of NeuroD1 [56]. In mice devoid of Mash1, a Cells severe loss was particularly found in neuronal precursors, with disappearance of the Notch signaling target Hes5, in It is noteworthy that stable overexpression of Slc38a1 not the ventral telencephalon enriched of Mash1 during neuro- only promoted proliferation for self-replication, but also genesis [57]. Notch signaling has been shown to mediate facilitated neuronal commitment, in undifferentiated P19 a cell–cell interaction to maintain dividing cells for sub- cells [52]. In undifferentiated P19 cells, constitutive expres- sequent generation of different progeny cell lineages in a sion is invariably found for transcripts of a variety of basic manner dependent on the expression of the repressor type helix-loop-helix (bHLH) transcription factors, which are bHLH genes Hes1 and Hes5, which both antagonized the all responsible for the positive and negative regulation of activator type neuronal bHLH factors such as Mash1 [58]. the functional state and differentiation trend in undifferen- However, both Hes1 and Hes5 were essential Notch effec- tiated NSCs [53]. These include activator (Mash1, Math3 tors in negatively regulating mammalian neuronal dif- and NeuroD1) and repressor (Hes1 and Hes5) types of ferentiation [59]. Accordingly, the drastic upregulation of bHLH factors. Stable overexpression of Slc38a1 drasti- both activator and repressor types of bHLH genes would cally up-regulated transcript expression of both the acti- be highly involved in molecular mechanisms underlying vator type Mash1, Math3 and NeuroD1 and the repressor the promotion of both proliferation and neuronal commit- type Hes5 without affecting that of Hes1 [52]. Cells were ment in pluripotent P19 cells with stable overexpression of then transfected with a luciferase reporter plasmid linked Slc38a1. to the NeuroD1 promoter from −1000 to +11 bp upstream Nevertheless, the mechanism how stable overexpres- for evaluation of the underlying mechanism of upregula- sion of Slc38a1 leads to upregulation of bHLH genes is not tion. A drastic increase was seen in the luciferase activity clarified so far. One possible but hitherto unproven specula- in undifferentiated stable Slc38a1 transfectants, whereas tion is that the intracellular level of l-GLN would be partly no significant change was found in the luciferase activity responsible for the promotion of transactivation of par- with deleted promoter constructs of either −366/+11 bp or ticular target genes such as bHLH transcription factors in −112/+11 bp upstream even in these stable transfectants. the nucleus of neural progenitor cells. l-GLN is shown to Although l-theanine was effective in drastically stimulating deteriorate the activation by l-arginine and l-leucine of the the promoter activity of NeuroD1 after sustained exposure mammalian target of rapamycin (mTOR) phosphorylation

1 3 Neurochem Res signals in rat intestinal epithelial cells [60]. By contrast, l- with a luciferase reporter plasmid of the Slc38a1 pro- 3 GLN inhibited the transport of [ H]l-arginine in cultured moter after sustained exposure to l-theanine. Although murine cerebellar and cortical neurons, in addition to sev- reporter plasmid analysis clearly revealed the requirement eral l-arginine analogues with a property to inhibit nitric of the promoter region between −1626 and −768 bp for the oxide synthase required for the formation of nitric oxide upregulation of Slc38a1 expression in pluripotent P19 cells from l-arginine [61]. l-arginine efflux was indeed medi- with sustained exposure to l-theanine [66], the mechanism ated in exchange for l-GLN influx through a mechanism underlying upregulation by l-theanine has yet to be clari- relevant to the transporter y + LAT2 classified as a mem- fied. Since l-theanine seems to be incorporated through ber of cationic amino acid transporters expressed in the l-GLN transporters expressed at cell surfaces in the brain brain [62]. However, l-arginine was a weak inhibitor of [1], sustained competitive inhibition by l-theanine would 3 3 the accumulation of both [ H]l-theanine and [ H]l-GLN promote gene expression of the target transporter Slc8a1, in rat brain synaptosomes, than l-theanine and l-GLN [1]. as a compensatory upregulation phenomenon often seen in These previous findings are again unfavorable for the possi- supersensitivity and/or sensitization for numbers of mem- ble links between pharmacological properties of l-theanine brane receptors under sustained inhibition by antagonists. and particular l-GLU receptor subtypes associated with the In silico analysis revealed the presence of several respon- formation of nitric oxide in the brain. sive elements at the upstream promotor region from −198 Activation of mTOR signaling only occurred in the pres- to −26 bp of Slc38a1 gene [43]. These included activator ence of an extracellular l-GLN as an obligatory component protein-1 (AP1) and cyclic AMP responsive element bind- in Jarkat cells [63]. Increased intracellular l-GLN levels led ing protein (CREB). In fact, artificial overexpression of to facilitation of the influx of extracellular essential amino either AP1 or CREB invariably induced a drastic stimula- acids (EAA) such as l-leucine, in exchange for the efflux tion of relative promotor activity of Slc38a1 gene in C6 of intracellular l-GLN, which consequently induced activa- glioma cells [43]. The transcription factor AP1 and/or tion of the mTOR signaling pathway required for protein CREB would at least in part play a role in the molecular translation, cell growth and deteriorated autophagy toward mechanisms underlying upregulation of Slc38a1 expression acceleration of cell growth and proliferation [64]. In fact, in pluripotent P19 cells exposed to l-theanine for a long intracellular l-GLN levels were more than doubled in P19 period. cells with stable overexpression of Slc38a1 than in cells Overexpression of X-box binding protein-1 (XBP1) with a mock vector [52]. Neural progenitor status was under predominantly led to a drastic increase in luciferase activ- the control of mTOR phosphorylation signals through a ity in P19 cells with the full length Slc38a1 reporter plas- mechanism related to the upregulation of Hes5 and Pax6 mid amongst a variety of expression vectors tested [66]. expression in pluripotent P19 cells [65]. These previous The reporter analysis showed the possible involvement findings all give rise to an idea that stable overexpression of a transcription factor related to endoplasmic reticu- of Slc38a1 would lead to increased intracellular l-GLN lev- lum (ER) stress relevant to XBP1 upregulation in the els and hence activation of the mTOR signaling pathway upregulation of Slc38a1 expression. The possibility that required for subsequent promotion of gene transactivation sustained exposure to l-theanine induces an ER stressor of particular bHLH transcription factors in NSCs. after the incorporation into the cytoplasm across membrane l-GLN transporters including Slc38a1, in associa- Activation by l‑theanine of Slc38a1 Gene Transcription tion with affecting intracellular levels ofl -GLN, is thus not ruled out. The ER stress was highly associated with To analyze the underlying mechanism for upregulation upregulation of XBP1 expression [67, 68] in neural pro- by l-theanine, pluripotent P19 cells were again used for genitor cells, while l-GLN attenuated ER stress and con- determination of the promoter activity for Slc38a1 gene sequential apoptosis in the colons in vivo and in vitro expression using luciferase reporter plasmids with different [69]. It is thus conceivable that l-theanine exacerbates lengths from −2959 to −768 bp upstream [66]. In pluripo- ER stress through a mechanism associated with reduced tent P19 cells exposed to l-theanine for a short period, no intracellular levels of l-GLN capable of scavenging ER significant increase was seen in the size of clustered pro- stress. l-GLN is shown to play a critical role in cell proliferating cells in contrast to the drastic increase in P19 liferation in a variety of cell types such as lymphocytes, cells exposed to l-theanine for a long period. Significant enterocytes and tumor cells [70]. In Caco-2 cells, l- increases were similarly found in the cluster size and MTT GLN increased proliferation activity through facilitation reducing activity in P19 cells cultured with l-theanine for a of nucleotide synthesis [71]. Nevertheless, the fact that long period, but not in those for a short period [66]. Lucif- sustained exposure to l-GLN itself failed to significantly erase activity was more than doubled in a manner sensitive promote neurosphere growth even at considerably high to the deletion of promoter regions in P19 cells transfected concentrations in contrast to l-theanine [66] argues in

1 3 Neurochem Res favor of an idea that upregulation of Slc38a1 expression Promotion of Adult Neurogenesis is a critical factor for inducing promotion of neurosphere growth and neuronal specification in neural progenitor To confirm pharmacological properties ofl -theanine in cells exposed to l-theanine for a long period. An alterna- adult neurogenesis besides embryonic neurogenesis, hip- tive interpretation is that sustained exposure to l-GLN at pocampal neural progenitor cells were isolated from young high concentrations does not markedly increase the intra- adult mice with predominant overexpression of green flu- cellular levels of this amino acid at concentrations effec- orescent protein (GFP) in cells expressing nestin [78]. In tive for upregulation of Slc38a1 expression as seen after hippocampal sections dissected from these adult Nestin- sustained l-theanine exposure. GFP transgenic mice, GFP fluorescence was predominantly detected in the subgranular zone known to be enriched of NSCs expressing nestin below the granular cell layer Promotion of Embryonic Neurogenesis stained with Hoechst33342 [76]. Prolonged culture with l-theanine markedly increased the size of neurospheres To exclude the possibility that aforementioned pharmaco- formed from neural progenitor cells in the adult mouse hip- logical properties of l-theanine are only restricted to pluri- pocampus [76]. The fact that sustained exposure to l-thea- potent P19 cells originally derived from carcinogenic cells nine increased the size of neurospheres isolated from the with immortality, we further explored the effects ofl -thea- hippocampus of adult Nestin-GFP transgenic mice gives nine on proliferation and neuronal specification in neural support for the proposal that l-theanine promotes prolifera- progenitor cells isolated from embryonic rodent brains. We tion for self-replication during adult neurogenesis. already confirmed the validity of neural progenitor cells In adult mice, the fatal traumatic stress water immersion isolated from embryonic rat [72] and mouse [73] neocor- restrain stress [79] induced a variety of sustained bidirec- tex in terms of the abilities to proliferate for clustering and tional behavioral abnormalities, such as flashback, numb- to differentiate into neuronal and astroglial lineages during ing and hyper-arousal, which were all seen in patients with culture, in addition to highly positive immunoreactivity for posttraumatic stress disorder [80]. These bidirectional nestin, as seen with NSCs. In our previous studies, further- abnormal behaviors were ameliorated by a daily intraperi- more, fundamentally identical features were similarly seen toneal injection of several antidepressants [80], which were in both activities to proliferate and to commit to neuronal shown to be able to promote both proliferation and neuronal and astroglial lineages between neural progenitor cells from differentiation in neural progenitor cells during adult neuro- adult hippocampus [74, 75] and embryonic neocortex [73] genesis [81–83]. A transient decline of BrdU incorporation in mice. was found in proliferating cells located in the hippocampal Sustained exposure to l-theanine accelerated the growth dentate gyrus (DG) in adult mice with the aforementioned of round neurospheres composed of clusters of prolif- severe fatal stress, together with bidirectional behavioral erating cells and MTT reducing activity in a concentra- abnormalities related to numbing and hyper-arousal [80]. tion-dependent manner at 1–100 µM in undifferentiated l-theanine was effective in preventing the transient decline neural progenitor cells isolated from embryonic rat neo- in the number of total and clustered cells incorporating cortex [66]. In these progenitor cells previously exposed BrdU in the DG when given orally for 5 consecutive days to l-theanine, marked promotion was seen in spontane- even after the fatal stress [84]. From the data cited above, ous and induced commitment to a neuronal lineage with it appears that l-theanine is endowed to promote the neuro- deteriorated astroglial specification. Sustained exposure to genesis essential for the production of new mature neurons, l-theanine selectively up-regulated transcript expression of in addition to modulating a variety of features of existing Slc38a1 in undifferentiated rat neural progenitor cells with- mature neurons, in embryonic, developing and adult brains. out affecting that of several differentiation inducer receptors, cell adhesion molecules and neurotrophic factors [66]. Similarly marked upregulation by l-theanine was found Activation of mTOR Signaling in Slc38a1 transcript expression in undifferentiated neural progenitor cells isolated from embryonic mouse neocortex Phosphorylation Cascades after sustained exposure [66], in addition to promotion of cell growth and neuronal commitment [76]. In newborn Intracellular levels of l-GLN is shown to be a crucial rats with chronic oral intake of l-theanine in drinking water determinant responsible for the mTOR kinase signal- in vivo, moreover, object recognition memory was signifi- ing activity, which highly contributes to the stimulation cantly enhanced along with accelerated incorporation of of both protein translation and cell growth, along with 5-bromo-2′-deoxyuridine (BrdU) in the hippocampal gran- attenuation of autophagy, through a mechanism relevant ule cell layer [77]. to several membrane transporters for this amino acid in

1 3 Neurochem Res cultured HeLa cells [64]. Evidence is accumulating for Nevertheless, the exact mechanism by which l-theanine the mTOR pathway as an intracellular downstream sig- promotes the phosphorylation of key molecules for mTOR nal of extracellular essential amino acids in the literature signaling in place of l-GLN in undifferentiated neural pro- [85]. In contrast, stable overexpression of Slc38a1 led to genitor cells is not well clarified so far. Both l-theanine and marked promotion of cellular proliferation and neuronal l-GLN could be similarly incorporated into the cytoplasm differentiation activities in pluripotent P19 cells [52]. through particular l-GLN transporters other than Slc38a1 Similarly, l-theanine was effective in accelerating both expressed on cellular surfaces, but would elicit differential activities in control stable mock transfectants, but failed properties for activation of the intracellular mTOR kinase to further activate the promotion of both proliferation and signaling processes in the cytoplasm. l-theanine would be neuronal differentiation activities in stable Slc38a1 trans- more efficient than l-GLN in promoting the mTOR signal- fectants [52]. ing pathway in neural progenitor cells in a particular situa- In undifferentiated embryonic murine neural progeni- tion. l-GLN, but not l-theanine, is shown to be metabolized tor cells cultured with l-theanine for a long period [76], in through intracellular glutaminolysis processes to produce fact, drastically accelerated phosphorylation was seen in α-ketoglutarate, which in turn facilitates the intracellular both mTOR and its downstream proteins such as p70S6K signaling mediated by mTOR kinases in association with and S6, which were all key proteins for the mTOR kinase the essential amino acid l-leucine [86]. The possibility pathway responsive to different amino acid signals, with that l-theanine may modulate the mTOR signaling activ- p70S6K levels being unchanged [76]. In pluripotent P19 ity through a mechanism completely irrelevant to l-GLN in cells stably overexpressing Slc38a1 capable of transport- the cytoplasm is thus not ruled out so far. ing both l-GLN and l-theanine in the brain, marked phosphorylation was similarly found in mTOR, p70S6K and S6 Predominance to NSCs without alteration of the endogenous level of p70S6K in the absence of l-theanine [76]. l-theanine was highly effective The expression profile of Slc38a1 transcript gives sup- in accelerating the phosphorylation of mTOR, p70S6K and port for the possible predominance of the pharmacologi- S6 in a concentration-dependent manner in control mock cal action of l-theanine on properties of NSCs in the brain. transfectants as seen in neural progenitor cells, but failed to Amongst different isoforms of the system A transporters additionally promote the phosphorylation of these mTOR tested, Slc38a1 transcript was predominantly expressed signaling molecules in stable Slc38a1 transfectants [76]. in the hypothalamus, with slightly detectable expression Intracellular signals mediated by the mTOR pathway in bone, in murine discrete organs examined as show in are crucial for the immature primitive features through a Fig. 2. No detectable expression of Slc38a1 was seen in mechanism associated with upregulation of several repres- other organs including liver, muscle, pancreas and adipose sor bHLH genes such as Hes5, in undifferentiated P19 cells tissues. In contrast, Slc38a2 mRNA was rather ubiquitously [65]. l-GLN suppressed the activation by l-arginine and expressed in different organs tested with a progressively l-leucine of the mTOR signaling in rat intestinal epithelial decreasing order in hypothalamus, liver, bone, muscle, cells [60], while activation of the mTOR pathway was only brown adipose tissue, white adipose tissue and pancreas. seen in the presence of extracellular l-GLN as an inevita- Expression of Slc38a4 transcript was highly localized in ble component in cultured Jarkat cells [63]. An increase in liver, muscle and bone, but not in hypothalamus. In the intracellular l-GLN levels led to acceleration of the influx discrete brain regions, cerebellum exhibited the highest of extracellular EAA such as l-leucine, in exchange for the expression of Slc38a1 mRNA, followed by olfactory bulb, efflux of intracellularl -GLN, followed by stimulation of midbrain, hypothalamus, cerebral cortex, hippocampus stri- the mTOR signaling pathway responsible for protein trans- atum and medulla-pons in a descending order. lation, cell growth and reduced autophagy, toward facilita- Taking into consideration the unique expression profile tion of cell growth for proliferation [64]. In stable Slc38a1 of bHLH transcription factors endowed to modulate fea- transfectants, marked upregulation was indeed seen in both tures of NSCs, accordingly, l-theanine seems to rather pre- activator (Mash1, Math3, NeuroD1) and repressor (Hes5) dominantly affect neurogenesis attributed to the generation types of bHLH genes as described above [52]. Accordingly, of new neurons from NSCs through mechanisms associ- sustained exposure to l-theanine could lead to upregulation ated with upregulation of Slc38a1 expression necessary for of Slc38a1 gene expression toward stimulation of the phos- increased intracellular l-GLN levels as proposed in Fig. 3. phorylation cascades mediated by mTOR signals of par- Increased intracellular l-GLN levels would then lead to ticular target proteins required for the induction of bHLH activation of mTOR signals required for transactivation of genes capable of modulating both immature property and activator and repressor types of bHLH genes as described differentiation status in undifferentiated neural progenitor above. In this scheme, two predominant action sites should cells. be involved in the promotion by l-theanine of neurogenesis

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Fig. 2 Expression profiles of system A transporter isoforms. Each tissue was dissected from C57BL/6J mice at 8 weeks old for subsequent determination of mRNA expression of different l-GLN transporter isoforms. CB cerebellum, CC cerebral cortex, HC hippocampus, HT hypothalamus, MB midbrain, MT medulla-pons, OB olfactory bulb, ST striatum

as a compensatory response to the sustained decrease in intracellular l-GLN levels during exposure to the transporter competitor l-theanine for a long term is not excluded so far.

Future Directions

Marked improvement is reported in cognitive abilities assessed by double-blinded physicians in healthy elderly age-matched people given capsules of green tea powder enriched of l-theanine compared to those with nor- Fig. 3 Hypothetical mechanism for l-theanine in NSCs. Long-term mal green tea powder capsules after daily oral intake for a exposure to l-theanine would lead to upregulation of the l-GLN period longer than 7 consecutive months up to 12 months transporter Slc38a1 responsible for the incorporation of extracellular l-GLN, followed by increased intracellular levels of l-GLN and [87]. A prospective study revealed that daily intake of subsequent facilitation of exchange transport mediated by the l-GLN green tea led to less incidence of both dementia and mild antiporter Slc7a5/8 of intracellular l-GLN with extracellular EAA. cognitive impairment in elderly people over 60 years old Increased intracellular EAA levels could thus result in activation of than occasional green tea drinkers when surveyed for 3 the mTOR signaling pathway required for the upregulation of different bHLH transcription factors essential for modulation of neurogen- consecutive years in the same regional residence in Japan esis in NSCs [88]. l-theanine as well as green tea is often beneficial for the amelioration of malfunctions and abnormalities under different stressful conditions toward mental relaxation after in NSCs. The first aspect is predominant upregulation by oral intake in humans as described above. l-theanine could l-theanine of Slc38a1 expression in NSCs, while the sec- be thus an amino acid ingredient of edible drinks and foods ond point is predominant transactivation by l-theanine of consumed for decades as a seed candidate useful for the bHLH factors after activation of mTOR signals in NSCs. development of innovative nutraceuticals and/or dietary Although Slc38a1 is highly localized in the brain, however, supplements for the prophylaxis in relation to the life style the reason why sustained exposure is absolutely required betterment without remarkable side effects seen with most for the upregulation by l-theanine of Slc38a1 expression in drugs required for therapy and treatment. From a viewpoint NSCs remains to be elucidated in future studies. An absurd of improvement of the quality of life in humans, prophy- groundless speculation that Slc38a1 would be up-regulated laxis is undoubtedly much better than therapy in likely

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