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Inhibition of the Glutamine Transporter SNAT1 Confers Neuroprotection in Mice by Modulating the mTOR-autophagy System

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Inhibition of the Glutamine Transporter SNAT1 Confers Neuroprotection in Mice by Modulating the mTOR-autophagy System

Daisuke Yamada et al. Commun Biol.

Abstract

The pathophysiological role of mammalian target of rapamycin complex 1 (mTORC1) in neurodegenerative diseases is established, but possible therapeutic targets responsible for its activation in neurons must be explored. Here we identified solute carrier family 38a member 1 (SNAT1, Slc38a1) as a positive regulator of mTORC1 in neurons. Slc38a1 flox/flox and Synapsin I-Cre mice were crossed to generate mutant mice in which Slc38a1 was selectively deleted in neurons. Measurement of 2,3,5-triphenyltetrazolium chloride (TTC) or the MAP2-negative area in a mouse model of middle cerebral artery occlusion (MCAO) revealed that Slc38a1 deficiency decreased infarct size. We found a transient increase in the phosphorylation of p70S6k1 (pp70S6k1) and a suppressive effect of rapamycin on infarct size in MCAO mice. Autophagy inhibitors completely mitigated the suppressive effect of SNAT1 deficiency on neuronal cell death under in vitro stroke culture conditions. These results demonstrate that SNAT1 promoted ischemic brain damage via mTOR-autophagy system.

Keywords: Cell death in the nervous system; Neurodegeneration.

Conflict of interest statement

Competing interestsThe authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of Slc38a1 expression in mouse tissues. a mRNA copy numbers of systems A (Slc38a1, Slc38a2, and Slc38a4), N (Slc38a3 and Slc38a5), L (Slc7a5 and Slc7a8), and ASC (Slc1a5) in the whole brain. Total RNA was extracted from whole brain, and the mRNA copy number of each gene was quantified using qRT-PCR. Values were normalized to those of Actb (n = 3). b Comparison of Slc38a1 mRNA levels among mouse tissues. Total RNAs were extracted from the indicated tissues, and the mRNA levels of Slc38a1 were compared using qRT-PCR. Values were normalized to those of Actb. (n = 4–6) c Identification of SNAT1-expressing cells in the cerebral cortex. Double-immunohistochemical using antibodies against SNAT1 and NeuN (neuron marker); single-immunohistochemical staining using antibodies against S100β (astrocyte marker), or CD11b (microglial marker). Nuclei were counterstained with Hoechst 33342. Scale bars = 20 µm. (vWAT, visceral white adipose tissue; sWAT, subcutaneous white adipose tissue; BAT; brown adipose tissue)
Fig. 2
Fig. 2
Generation of neuron-specific Slc38a1 knockout mice. a Targeting strategy to create the floxed Slc38a1 allele (Slc38a1flox). Slc38a1 exon 2 is flanked by loxP sites. The flippase recombinase target-flanked Neo cassette was removed by crossing with CAG-FLP mice. Exon 2 was removed by crossing with SynI-Cre mice to selectively produce the Δ allele in neurons. b Southern blot analysis to confirm the recombination with the targeting vector at the genomic Slc38a1 locus. Genomic DNA from embryonic stem cells was digested with AfIII and hybridized with a DIG-labeled 3′ probe. c PCR analysis verifying the Δ allele in mutant mice. Genomic DNA was extracted from the brain of each indicated mouse, and PCR products derived from the wild-type, flox, or Δ allele were detected. d Quantification of Slc38a1 and Slc38a2 mRNA levels in whole brains of from mutant mice. Total RNAs were extracted from whole brains of control or mutant mice, and the mRNA levels of Slc38a1 and Slc38a2 were compared using qRT-PCR. Values were normalized to those of Gapdh (n = 3). e Deletion efficiency of Slc38a1 in brain segments. Proteins were extracted from each indicated brain segment of control or mutant mice, and SNAT1 was detected using western blotting. CBB staining was used as a loading control. C and M indicate control and mutant, respectively. f Confirmation of neuron-specific Slc38a1 deficiency in mutant mice. Double-immunohistochemical staining using antibodies against SNAT1 and NeuN. Nuclei were counterstained with Hoechst 33342. Scale bars indicate 100 µm
Fig. 3
Fig. 3
Neuron-specific Slc38a1 deficiency confers resistance to ischemic brain injuries. a, b TTC staining of a brain section prepared from MCAO model mice. Representative images of the coronal bregma section a and measurements of the infarct area at each indicated point from the bregma (b, n = 5). c, d Immunohistochemical detection of the neurodegenerative effect of MCAO on cerebral neurons. Brain sections at the bregma were stained with antibodies against the neuronal markers NeuN (c, n = 3) or MAP2 (d, n = 3), and areas with undetectable staining were measured
Fig. 4
Fig. 4
mTORC1 activation promotes ischemic brain injuries. a Assessment of mTORC1 activity in the brain after MCAO. Cerebral cortex samples were isolated from the contralateral or ipsilateral region at each indicated time point after MCAO, and the phosphorylation of p70S6k1 was compared by measuring the pp70S6k1(T389)/p70S6k1 ratio (n = 3). b mTORC1 activity in cerebral neurons after MCAO. Brain sections were prepared from mice 1 h after MCAO, and NeuN- or SNAT1 -positive cells in the cerebral cortex were double stained with an antibody against pp70S6k1(T389). Bars = 100 µm. c, d Inhibitory effect of neuron-specific Slc38a1 deficiency on mTORC1 activation by MCAO. Whole brain samples were isolated from control or mutant mice 1 h after MCAO, and pp70S6k1(T389) levels in the cerebral cortex were compared using immunohistochemistry c or western blotting (d, n = 3). Bars = 100 µm. e Suppression of the neuroprotective effect of Slc38a1 deficiency through Tsc1 heterozygosity. Whole brain samples were prepared from control, mutant, or mutant/Tsc1flox/wt mice after MCAO, and brain sections at the bregma were stained with an anti-MAP2 antibody to measure determine unstained areas (n = 3)
Fig. 5
Fig. 5
SNAT1 activates mTORC1 in cerebral neurons. a Procedure for deleting Slc38a1 using an in vitro culture system. Primary neurons isolated from the cerebral cortices of Slc38a1flox/flox mice were infected with a lentivirus encoding inactive ΔCre or active Cre, and control or Slc38a1-null neurons were prepared. b, c Deletion efficiency of Slc38a1 from primary neurons. Total RNA and protein were extracted from control or Slc38a1-null neurons, and the expression levels of Slc38a1 were assessed using qRT-PCR (b, n = 3) or western blotting (c, n = 3). df Analysis of mTORC1 activity after Slc38a1 deficiency. Proteins were extracted from control or Slc38a1-null neurons, and pp70S6k1(T389), pmTOR(S2448), and pS6(S235/236) were detected using western blotting. GAPDH served as a loading control (n = 3)
Fig. 6
Fig. 6
Autophagy is a critical mediator of neuroprotection conferred by Slc38a1 deficiency. a, b Assessment of the neuroprotective effect against ischemic stress conferred by Slc38a1 deficiency. Control or Slc38a1-null neurons were cultured under normal (anaerobic glucose deprivation [OGD]) or OGD conditions and stained with an anti-MAP2 antibody (a, n = 3) or PI (b, n = 6) to evaluate dead neurons. Bars = 100 µm. cg Increases in the mRNA levels of autophagy-related genes associated with Slc38a1 deficiency. Total RNA was extracted from control or Slc38a1-null neurons, and the mRNA levels of each indicated gene were measured using qRT-PCR. Values were normalized to those of Gapdh (n = 6). h Evaluation of autophagy under ischemia. Cell-free lysates of Slc38a1-null neurons cultured under OGD were subjected to western blotting using anti-phospho-p62 and anti-actin antibodies (n = 4). i, j Suppressive effect of autophagy inhibitors on neuroprotection induced by Slc38a1 deficiency. Control or Slc38a1-null neurons were treated with 1 nM bafilomycin i or 5 µM chloroquine j in the presence or absence of OGD. Neuronal cell death was assessed using PI staining (n = 3)

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