Therapeutic relevance of mTOR inhibition in murine succinate semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA metabolism
a b s t r a c t
Aldehyde dehydrogenase 5a1-deficient (aldh5a1−/−) mice, the murine orthologue of human succinic semialde- hyde dehydrogenase deficiency (SSADHD), manifest increased GABA (4-aminobutyric acid) that disrupts au- tophagy, increases mitochondria number, and induces oxidative stress, all mitigated with the mTOR (mechanistic target of rapamycin) inhibitor rapamycin [1]. Because GABA regulates mTOR, we tested the hypoth- esis that aldh5a1−/− mice would show altered levels of mRNA for genes associated with mTOR signaling and ox- idative stress that could be mitigated by inhibiting mTOR. We observed that multiple metabolites associated with GABA metabolism (γ-hydroxybutyrate, succinic semialdehyde, D-2-hydroxyglutarate, 4,5-dihydrohexanoate) and oxidative stress were significantly increased in multiple tissues derived from aldh5a1−/− mice. These meta- bolic perturbations were associated with decreased levels of reduced glutathione (GSH) in brain and liver of aldh5a1−/− mice, as well as increased levels of adducts of the lipid peroxidation by-product, 4-hydroxy-2- nonenal (4-HNE). Decreased liver mRNA levels for multiple genes associated with mTOR signaling and oxidative stress parameters were detected in aldh5a1−/− mice, and several were significantly improved with the adminis- tration of mTOR inhibitors (Torin 1/Torin 2). Western blot analysis of selected proteins corresponding to oxida- tive stress transcripts (glutathione transferase, superoxide dismutase, peroxiredoxin 1) confirmed gene expression findings. Our data provide additional preclinical evidence for the potential therapeutic efficacy of mTOR inhibitors in SSADHD.
1.Introduction
Inhibitory GABA derives from the primary excitatory neurotransmit- ter, glutamate, via decarboxylation catalyzed by glutamate decarboxyl- ase (GAD). GABA is metabolized to succinic acid via GABA-transaminase (GABA-T; Fig. 1) and succinic semialdehyde dehydrogenase (ALDH5A1; aldehyde dehydrogenase 5a1 = succinic semialdehyde dehydrogenase (SSADH)). Mendelian disorders of both GABA-T and ALDH5A1 exist [2–4]. ALDH5A1 deficiency (also referred to as succinic semialdehyde dehydrogenase deficiency (SSADHD)) presents with a non-specific neu- rological phenotype encompassing developmental delay, expressive language impairment, variable epilepsy [5,6], and neuropsychiatric problems (i.e. ADHD, OCD, aggression). The biochemical hallmarks ofSSADHD include accumulation of glutamate, GABA and the GABA- derivative γ-hydroxybutyrate (GHB) [7] in physiological fluids, the lat- ter an intermediate with a complex pharmacology and the cardinal physiological biomarker of SSADHD [8] (Fig. 1). Our understanding of the pathophysiology of SSADHD has evolved from studies in the murine model [9]. This model was derived by standard cre-lox recombination, with deletion of exon 7 containing a catalytic cysteine residue [10]. Metabolically, aldh5a1−/− mice represent a valid phenocopy of the human disease, with elevations of both GABA and GHB in tissues and biofluids [11]. Aldh5a1−/− mice manifest a seizure phenotype, including progression from absence to tonic-clonic seizures to lethal status epilep- ticus by day of life (DOL) 25 [12]. Accordingly, metabolic or other studies, as well as therapeutic considerations, must be implemented within the first 2–3 weeks of life.Lakhani and coworkers recently defined a role for GABA in autophagy in yeast. High levels of GABA impaired both mitophagy and pexophagy [1].
These findings were reproduced in aldh5a1–/– mice. In- tervention with the mTOR inhibitor rapamycin resulted in normaliza- tion of elevated hepatic pS6 (a kinase linked to mTOR function), superoxide dismutase (SOD), and mitochondrial numbers. Comparable impairments of autophagy have been identified associated withvigabatrin intervention, an antiepileptic employed for treatment of in- fantile spasms which irreversibly inactivates GABA-T and elevates GABA [13]. Neurophysiological data also suggests synergistic actions be- tween GABA and mTOR. For example, mTOR mediates synaptic regula- tion through modulation of miniature inhibitory postsynaptic currents that are GABA-mediated [14], and hyperactive mTOR elevates evoked synaptic responses in both glutamatergic and GABAergic neurons. Moreover, mTOR may be activated by calcium signaling mediated by GABAB receptors [15]. The preceding observations suggest that mTOR is an appropriate therapeutic target for diseases with altered GABA homeostasis.Here, we extend our characterization of mTOR inhibitors as a therapeutic consideration for SSADHD, employing aldh5a1−/− mice as the experimental platform. Initially, we characterized elevations of GABA-associated metabolites in brain, liver and kidney of aldh5a1−/− mice vs. aldh5a1+/+ mice, including GABA, GHB, D-2- HG, SSA, and DHHA (see Fig. 1). The majority of these metabolites in- duce oxidative stress when administered individually to rodents [16–20]. We then confirmed significant depletion of reduced gluta- thione (GSH) in brain and liver of aldh5a1−/− mice [16], and extend- ed those findings by documenting significant depletion of total GSH in extracts of RBCs (red blood cells) derived from patients with SSADHD [21]. Additionally, we demonstrated elevated 4-HNE conju- gates in extracts of brain derived from aldh5a1−/− as compared to aldh5a1+/+ mice, a relevant finding since SSADH is the primary alde- hyde dehydrogenase responsible for further oxidation of 4-HNE in rodent brain [22]. These metabolic evaluations set the stage for a novel, comprehensive characterization of mRNA levels associated with genes involved in both mTOR signaling and the maintenance of intracellular oxidation-reduction potential, and the potential ben- eficial effect of agents which inhibit mTOR signaling (Tor 1/Tor 2) on these mRNA levels and selected related proteins.
2.Methods
All animal procedures were approved by the Washington State Uni- versity IACUC (protocol 0432 and 0476). Aldh5a1−/− mice were gener- ated via harem breeding (aldh5a1+/− females (2) x aldh5a1+/− male). This strain, developed in the Gibson laboratory, is syngeneic on the C57Bl background, and has been backcrossed to wild-type C57Bl stock yearly for 10 years. The model is a complete null for aldh5a1 activity due to the loss of exon 7 containing an active site cysteine required for activity [10]. At birth, offspring are subjected to tail snip for production of genomic DNA. The latter is employed for nested 3-primer PCR genotyping [10]. All mice were maintained on a standard 12 hour light/dark schedule, with ad-libitum delivery of standard mouse chow (Teklad 2018, Harlan Laboratories). Mice were sacrificed by 1-2 minute exposure to carbon dioxide followed by cervical dislocation. Tissues were immediately dissected on an ice-cold glass plate followed by rapid immersion into dry ice/ethanol and storage at − 80 °C. Whole blood was obtained from patients with SSADHD and control individuals with informed consent (IRB 12678). RBCs were isolated by centrifuga- tion on Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO, USA), and pelleted RBCs washed with PBS and stored at −80 °C until analysis.Torin 1 and 2 were obtained from Tocris Biosciences (Bristol, United Kingdom), and were dissolved in DMSO at 25 mg/mL. Stock solutions were diluted in PBS and administered to mice via intraperitoneal injec- tion commencing at day of life (DOL) 10. Antibodies were purchased from ThermoFisher Scientific (Waltham MA) except GAPDH (Santa Cruz Biotechnologies; Santa Cruz CA). The final DMSO percentageadministered to animals was less than 0.1%. Both aldh5a1+/+ and un- treated aldh5a1−/− mice received vehicle control injections.Metabolites quantified in liver, kidney and brain derived from aldh5a1−/− and aldh5a1+/+ mice included GHB, GABA, succinic semialdehyde (SSA), D-2-hydroxyglutaric acid (D-2-HG), and 4,5- dihydroxyhexanoic acid (DHHA) (Fig. 1). Animals were 17–20 days of age, and of mixed gender.
Metabolites were quantified in tissues stored at −80 °C until work-up. Tissues were homogenized at 1:10 w/v in 3% sulfosalicylic acid and supernatants obtained by centrifugation. Tissues were harvested from n = 8 each aldh5a1+/+ and aldh5a1−/− mice that were age-matched littermates of mixed gender. Metabolite quantitation in tissue supernatants was performed using isotope dilution methodolo- gy. GHB was measured using gas chromatography-mass spectrometry (GCMS) and 2H6-GHB as internal standard [23]. GABA was measured by electron-capture negative-ion mass fragmentography employing 13C2- GABA as internal standard [24]. SSA was quantified as the dinitrophenylhydrazine derivative employing liquid chromatography- tandem mass spectrometry (LC-MS/MS) and 13C4-SSA as internal stan- dard [25]. D-2-HG was quantified as described [26] using chiral separa- tion. In brief, D-2-HG was separated from L-2-hydroxyglutaric acid employing stable-isotope-dilution liquid chromatography-tandem mass spectrometry following derivatization with diacetyl-L-tartaric anhydride. The method for quantitation of DHHA was similar to that for GHB and employed (2H3-CH3)-4,5-DHHA (synthesized in-house) as internal stan- dard [27]. Metabolite concentrations were standardized to wet weight of tissue. Metabolic analyses were performed in whole organs.GSH and 4-HNE adducts were quantified in brain and liver obtained from n = 8 each aldh5a1+/+ and aldh5a1−/− mice that were age- matched littermates of mixed gender. GSH is the major tripeptide intra- cellular antioxidant, while 4-HNE represents an α,β-unsaturated hydroxyalkenal produced during cellular lipid peroxidation, the primary unsaturated hydroxyalkenal formed during lipid peroxidation. 4-HNE is short-lived due to the presence of 3 reactive groups, including aldehyde, double-bond and hydroxyl-moiety (Fig. 1).Reduced GSH was quantified using a modification of the procedure by Moore and coworkers [28].
RBCs derived from patients with SSADHD (n = 9) and control indi- viduals (unaffected siblings of patients, parents and unrelated controls; n = 7, not age- or gender-matched) were isolated with standard Ficoll gradient centrifugation and quantified for oxidized, reduced and total GSH by colorimetric assay obtained from Trevigen (Gaithersburg, MD) using manufacturer’s suggested protocol.Adducts of 4-HNE were quantified in brain and liver derived from n = 8 each aldh5a1+/+ and aldh5a1−/− mice that were age-matched lit- termates of mixed gender. The protocol employed a colorimetric ELISA assay from CellBiolabs (San Diego, CA). A BSA-4-HNE standard contained in the ELISA kit was employed to obtain standard curves for quantitation.RNA was prepared by pooling liver/brain tissues (whole organs) with n = 4 for wild-type (WT) and mutant (MUT) mice, day oflife (DOL) 21, n = 2 survivors for Tor1 (5 mg/kg daily; DOL 50); and n = 1 survivor for 5 mg/kg Tor2 (DOL 50). Samples were homogenized with TRIzol® (Invitrogen). Samples for Tor 1 and 2 were taken from aldh5a1−/− mice that were 50 days old, which is highly significant in view of uniform lethality by day of life (DOL) 25 in the absence of ther- apy [10,29]. Accordingly, parallel untreated aldh5a1−/− mice were not available for comparison to treated mice at the DOL 50 timepoint. Ho- mogenates were chloroform extracted, and RNA was precipitated with isopropanol and ethanol followed by brief air drying and reconstitution in water. RNA was quantified via absorbance and purity assessed using 260 nm/280 nm absorbance ratio (N 1.8). iScript mastermix (Biorad, Hercules CA) was employed to generate cDNA employing an Eppendorf mastercycler per manual instructions with 1 μg RNA/20 μL reaction. Sybr green (Biorad) reactions were prepared as a master mix with 50 ng of cDNA/10 μL/well pipetted into prearrayed and validated (per MIQE guidelines; [30]) mTOR pathway analysis plates and oxidative stress arrays (M394 PrimePCR™ SABioscience).
A BioRad CFX384 real- time PCR with BioRad CFX manager v3.0 software was employed for data acquisition, normalization, expression and statistical analysis with significance set at p b 0.05 based upon two replicates per group. Statistical analysis for the arrays employed a one-way ANOVA with post-hoc Bonferroni t-test correction, as described [31]. Aldh5a1−/− mice (treated or untreated) were compared to the expression level de- tected for untreated, control aldh5a1+/+ mice (expression set to 1.0). The Biorad website describes for the prime PCR how the relevant genes have been compiled for expression arrays: http://www.bio-rad. com/en-us/prime-pcr-assays/pathway/primepcr-pathways.Liver samples (whole organs) were mechanically homogenized in cold RIPA buffer with antiprotease and antiphosphatase tablets (Roche, Pleasanton CA), sonicated for 5 min in an ice bath, and quanti- tated for total protein using the Pierce bicinchoninic acid (BCA) colori- metric protein assay (Abs 562 nm). Samples and BSA standards were read on a BioTek Synergy 4 microplate reader and extrapolated with GraphPad Prism 6. Sample homogenates were diluted in PBS, lithium dodecyl sulfate (LDS) sample buffer and 50 mM dithiothreitol (DTT) re- ducing agent to a loading concentration of 50 μg/well and incubated at 70 °C for 10 min, vortexed, and placed immediately on ice. 30 μL was loaded into Bolt 4-12% Bis-Tris pre-cast polyacrylamide gels and electro- phoresed for 35 min at a constant 165 volts in MES running buffer. Pro- tein was transferred to nitrocellulose using the iBlot2® transfer system, blocked, and probed with alkaline phosphatase conjugated secondary antibody. Protein bands were detected using chemiluminescent CDP- Star® Substrate with Nitro-II™ Enhancer (Novex) on a BioRad gel doc- umentation imager and analyzed by normalization to either GAPDH or vinculin (loading controls).
3.Results
Concentrations of GHB, GABA, SSA, D-2-HG and DHHA in extracts of brain, liver and kidney were obtained for aldh5a1+/+ and aldh5a1−/− mice (Fig. 2A–E). Values were (all nmol/mg tissue, n = 8 mice of each ge- notype, mean ± SEM, aldh5a1−/− mice and aldh5a1+/+ mice in parenthe- ses): GHB in liver, 481 ± 133 (5.7 ± 0.8); kidney, 514 ± 68 (12 ± 2.5);brain, 510 ± 65 (1.9 ± 0.2); GABA in liver, 510 ± 46 (31 ± 4); kidney,651 ± 14 (54 ± 16); brain, 8915 ± 167 (2109 ± 37); SSA in liver,15 ± 1.4 (5.4 ± 0.5); kidney, 21 ± 1.4 (14 ± 0.6); brain, 23 ± 3(2.8 ± 1.2); D-2-HGA in liver, 1857 ± 213 (18 ± 4); kidney, 1191 ±101 (37 ± 2.8); brain 85 ± 7.5 (28 ± 1.3); DHHA in liver, 5.7 ± 0.6(none detected); kidney, 56 ± 8.3 (0.5 ± 0.2); brain, 80 ± 9.6 (0.2 ± 0.2). Concentrations of all metabolites were significantly increased intissues of aldh5a1−/− mice as compared to aldh5a1+/+ (p b 0.01, two tailed t test).Reduced glutathione (GSH) was quantified in extracts of brain and liver derived from aldh5a1−/− and aldh5a1+/+ mice (n = 8 each genotype, mixed gender, age-matched, aldh5a1+/+ with aldh5a1−/− mice in parentheses): brain, 102 ± 7 (67 ± 9, mean ±SEM, values reported as PAR (peak area ratio in relation to stable- isotope labeled internal standard); liver, 156 ± 12 (95 ± 14) (Fig. 3A; p b 0.01 for both brain and liver, two-tailed t test). We next examined the level of total GSH in RBCs derived from patients with SSADHD (n = 9) and control individuals (n = 7, composed of parents of patients, unaffected siblings, and unrelated control indi- viduals, mixed gender and not age-matched).
Total GSH was 330 ± 13 nmol/ml packed RBC (red blood cells) for control individuals (mean ± SEM) and 273 ± 22 for SSADHD patients (p b 0.05, two tailed t test with Welch’s correction) (Fig. 3B).Because ALDH5A1 is the major aldehyde dehydrogenase responsible for further metabolism of 4-HNE [22], we tested the hypothesis that adducts of this reactive aldehyde intermediate would be elevated in brain and liver of aldh5a1−/− mice. Free 4-HNE is a very short-lived species, and its three reactive sites make it a target for adduction of var- ious species (e.g., GSH). For 4-HNE adducts, we found in brain of aldh5a1+/+ mice 33.6 ± 1.8 μg/mg protein (n = 8 mice, mean ± SEM, mixed gender) and in aldh5a1−/− mice 48.1 ± 6.2 (p b 0.05, two tailed t test) (Fig. 3C). These values in liver extracts were 30.5 ± 0.7 for aldh5a1+/+ and 39.5 ± 5.7 for aldh5a1−/− mice (p = ns, two tailed t test).Based on earlier studies of the effect of rapamycin on mTOR signaling and biomarkers of oxidative stress in aldh5a1−/− mice [1], we next in- vestigated the patterns of gene expression at the mRNA level for both aldh5a1+/+ and aldh5a1−/− mice, with a focus on genes involved in mTOR signaling and maintenance of the intracellular oxidation- reduction homeostasis. Further, we examined the capacity of newer generation drugs (Tor 1, Tor 2) that inhibit mTOR signaling to improve mRNA levels corresponding to these genes (Tables 1 and 2). Additional rationale for examining the molecular aspects (mRNA levels) related to Tor 1 and Tor 2 intervention in aldh5a1−/− mice derived from our find- ing that these agents are capable of significantly extending the lifespan of these mice [32].
In liver, the direction of expression of mRNA was decrease in un- treated aldh5a1−/− mice for 13 of 20 genes quantified, as opposed to in- crease of mRNA levels for 7 genes. Either Tor 1 and Tor 2 resulted in the normalization (not significantly different from aldh5a1+/+ mice) of mRNA levels for Akt1a1, Ddit4, Eif4e and Rps6ka1 (Table 1). The applica- tion of Tor 2 resulted in the normalization of mRNA levels in aldh5a1−/− mice for Pten, Rhoa, Rraga, Sgk1 and Tsc 1 (see Table 1 for quantitation ofLegend: RNE, relative normalized expression; for each gene, the first row represents un- treated aldh5a1−/− mice, the middle row aldh5a1−/− mice treated with Torin 1, and the final row aldh5a1−/− mice treated with Torin 2.mRNA levels). Overall, the use of Tor 1 and 2 was associated with normalization of mRNA levels in aldh5a1−/− mice for 50% of genes (10/20) which showed dysregulated mRNA levels in aldh5a1−/− mice (untreated) compared to aldh5a1+/+ mice. Conversely, looking at themRNA level of mTOR-associated genes in the brain (23 total genes), re- sults were less impressive than results in the liver. In brain, the direction of mRNA level was decrease in untreated aldh5a1−/− mice for 13 of 23 genes quantified, as opposed to increase of mRNA level for 10 genes (data not shown). Only the mRNA level of Akt1s1 in aldh5a1−/− mice was normalized to levels not significantly different from aldh5a1+/+ mice by treatment with Tor 2 (data not shown). A schematic diagram of the relationship of the majority of these genes (Table 1), and their re- lationship to mTOR signaling, is shown in Fig. 4.
We next turned attention to profiling of mRNA levels in adlh5a1+/+ and aldh5a1−/− mice, and for the latter those that had been rescued from premature lethality with Tor 1 and 2, that focused on oxidative stress and/or maintenance of the intracellular redox potential in the liver. This followed on our earlier work in which we had seen normali- zation of superoxide dismutase protein levels, and enzyme activity, in the liver of aldh5a1−/− mice treated with rapamycin [1]. In terms of mRNA levels reflecting expression of genes indicative of oxidative stress (Table 2), the level of mRNA in untreated aldh5a1−/− mice was a de- crease in Akr1c18, Aldh1a1, Casp3, Snca, as opposed to increase in mRNA levels for Ddit3 and Dusp1) (Table 2). Treatment of aldh5a1−/− mice with Tor 1 normalized the mRNA levels for Dusp1 to a level indis- tinguishable from levels in aldh5a1+/+ mice. Conversely, treatment of aldh5a1−/− mice with Tor 2 resulted in the normalization of mRNA levels for Aldh1a1, Casp3, and Ddit3 to levels not significantly different from aldh5a1+/+ mice. The mRNA levels for genes representing antiox- idant enzymes in the liver was somewhat different, revealing that thedirection of mRNA level for the 15 measured genes was almost uniformly decrease in untreated aldh5a1−/− mice as compared to aldh5a1+/+ mice (Table 2; 14/15 genes overall). Intervention in aldh5a1−/− mice with ei- ther Tor 1 and Tor 2 resulted in mRNA levels of Sod1 and Prdx2 that were indistinguishable from those observed for aldh5a1+/+ mice.
Intervention in aldh5a1−/− mice with Tor 2, however, resulted in mRNA levels for Cat, Sod3, Gpx3, and Prdx6 (Table 2) to levels that were not significantly different from the levels observed in aldh5a1+/+ mice.To verify the results of gene expression analyses, we undertook im- munoblot analysis of selected proteins in liver of aldh5a1−/− mice at two ages, DOL 11 and DOL 20 (Fig. 5). At both ages, aldh5a1−/− mice demonstrated a significant decrease of GSTT1 (glutathione transferase theta 1 subunit; Fig. 5B, F), consistent with down-regulated levels of mRNA (Table 2). Similar results were observed for the dimeric form of PRDX 1 (peroxiredoxin 1; Fig. 5D) which is the active form of the protein, accompanied by an increased concentration in DOL 20 aldh5a1−/− mice of the inactive monomeric form (Fig. 5H) [33]. SOD2 (superoxide dismutase 2, mitochondrial located) was elevated in aldh5a1−/− mice at both ages, although only significantly at DOL 11 (Fig. 5C, G). This is consistent with earlier work showing increased SOD2 enzyme activity in this mouse model, and reflects the elevated mitochondrial number in these mice which was previously improved with rapamycin [1]. Since aldh5a1 is ablated in these animals, we ex- plored several additional aldehyde dehydrogenase isoforms (aldh1a1,aldh2, aldh 3a1, aldh4a, aldh6a1, aldh7a1 and aldh9a1), in order to ex- plore potential compensation in the mouse model for loss of aldh5a1. We found decreased levels of the 3a1 species and increased amounts of the 6a1 species (Fig. 5A, C). Of interest, aldh3a1 has been suggested to promote resistance to 4-HNE-induced oxidative damage in the cor- nea, and lowered levels of this protein may add to the level of oxidative stress in aldh5a1−/− mice [34]. Aldh6a1 encodes methylmalonate sem- ialdehyde dehydrogenase, an enzyme on the valine catabolic pathway, with mitochondrial location [35,36]. The mitochondrial location of this protein, and its elevated levels, is consistent with data on SOD2 in rela- tion to increased mitochondrial numbers in aldh5a1−/− mice [1].
4.Discussion
The current report extends our characterization of mTOR inhibitors as a therapeutic consideration for SSADHD. We present the first comprehen- sive analyses of GABA, GHB, D-2-HG, SSA, and DHHA (Fig. 2A–E) in mul- tiple tissues, also demonstrating for the first time a significant increase of SSA in peripheral tissues of aldh5a1−/− mice [16,27,29,37]. The majority of these metabolites induce oxidative stress when administered individu- ally to rodents [16–20], which formed the rationale underlying our gene expression analysis targeting the cellular oxidation-reduction potential. The data of Fig. 2 suggests that the metabolic equilibrium in aldh5a1−/− mice favors GHB production from SSA by ~25–30-fold. Why SSA levels would be present in aldh5a1+/+ mice, in view of the low Km of SSADH (~1–4 μΜ) [38] for SSA, is not readily explained. The presence of SSA in aldh5a1+/+ might relate to the “dynamic catalytic loop” of the SSADH protein which is susceptible to oxidative stress conditions and subse- quent inactivation [39–41], suggesting that some level of SSA might al- ways be present. Of interest, a recent report has demonstrated that aldh1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons [42]. The finding of significantly decreased mRNA encoding aldh1a1 (Table 2) could potentially help to explain the increased levels of SSA in aldh5a1−/− mice (combined with loss of aldh5a1), but does not readily shed light on the accumulation of SSA in aldh5a1+/+ mice.
The data of Fig. 3 verify global disruptions of the cellular oxidation- reduction potential in aldh5a1−/− mice, and we verify for the first time that total glutathione is significantly depleted in multiple patients with SSADHD, and 4-HNE adducts are significantly increased in the liver of aldh5a1−/− mice. Our rationale for evaluation of 4-HNE adducts cen- tered on our earlier work verifying that SSADH is the major enzyme in- volved in 4-HNE metabolism in brain [22], but not in liver, and the prediction with the loss of SSADH activity in aldh5a1−/− mice that 4- HNE, and correspondingly 4-HNE adducts, would be increased. Increased levels of 4-HNE in the brain of aldh5a1−/− mice, and by ex- trapolation to the brain of patients with SSADHD, might correlate with the clinical picture of patients, in which neuroimaging reveals hyperintensities of the basal ganglia [2]. These hyperintensities may well relate to oxidative stress [43]. Armed with the preceding metabolic evidence for oxidative stress in aldh5a1−/− mice, we opted to pursue a global assessment of both mTOR signaling and oxidative stress employing gene expression analysis for mRNA levels. Our rationale for these studies centered on earlier work with GABA, mTOR and oxidative stress [1,13,32], in which we showed that elevated GABA, acting via mTOR, induced accumulation of mitochondria and enhanced oxidative stress. The finding that rapamycin could mitigate these effects led us to investigate newer generation mTOR inhibitors. We chose to utilize Torin 1 in lieu of rapamycin because Torin 1 is highly potent and selec- tive, and several mTORC1 (mechanistic target of rapamycin complex 1) functions are resistant to inhibition by rapamycin, yet effectively blocked by the newer analog Torin 1 [44,45]. Torin 2 is a novel, second-generation ATP-competitive inhibitor that is potent and selec- tive for mTOR with a superior pharmacokinetic profile to previous in- hibitors, including Torin 1 [46]. Accordingly, we evaluated the treatment of aldh5a1−/− mice with both compounds, followed by sub-sequent studies of mRNA levels.
The data of Table 1, focused on mRNA levels in aldh5a1−/− mice in the presence and absence of mTOR inhibitors, provide further evidence for abnormal PI3K/AKT/mTOR signaling (Fig. 4). Concentrations of mRNA encoding effectors of this signaling pathway were clearly dysregulated in untreated aldh5a1−/− mice, including decreased levels of mRNA encoding Ulk2 and Elf4e, and increased mRNA levels encoding Rps6ka1 (Table 1). These expression anomalies were not induced by a corresponding increase in mRNA levels for insulin-like growth factor (Igf-1), whose mRNA levels were actually decreased in ald5a1−/− mice. The relationships between IGF-1 and mTOR signaling have been well established [47]. Consistent with our finding of low Igf-1 mRNA in aldh5a1−/− mice was the demonstration of low Igf1 levels in blood from a patient with GABA-transaminase deficiency [48], a disorder also accompanied by elevated GABA in tissues and body fluids. Auto- crine GABA (working via activation of GABAA receptors) can depolarize pancreatic β-cells and enhance insulin secretion, and insulin can down- regulate GABAA-ergic signaling as a feedback mechanism for fine- tuning of insulin secretion [49,50]. Importantly, mRNA levels encoding Sgk1 were increased in untreated aldh5a1−/− mice, and Sgk1 is a gene whose activity is induced by both PI3K and mTORC2. Dysregulation of the mRNA levels encoding Sgk1 has been associated with oxidative stress, as well as dopamine maintenance and epilepsy, all of which form components of the underlying SSADHD phenotype.
Previous work from our lab and others adds further support to the probable relationship between elevated GABA and altered mTOR expression seen in Table 1. Along those lines, the data of Lakhani and coworkers (2014) verified that increased mTOR signaling, as measured using phosphoS6 kinase analysis, could be significantly attenuated with the mTOR inhibitor rapamycin. These studies were performed both in brain and liver, which verified a more systemic correction that lends further support to our expression array data. Additionally, inhibition of mTOR leads to mRNA degradation of selected species in brain while mTOR still regulates the translation of selected mRNAs [51]. These obser- vations may underscore why we saw limited (if any) correction of expres- sion analyses in the brain of aldh5a1−/− as opposed to the significant corrections observed in liver.Turning attention to Table 2 and mRNA levels associated with oxida- tive stress, previous studies in aldh5a1−/− mice have shown that total radical-trapping antioxidant potential was significantly decreased in liver and cerebral cortex, SOD (superoxide dismutase) activity was elevated in liver and cerebellum, and catalase activity (a quencher of peroxisomal hydroxyl radicals) was elevated in thalamus [17,18]. Addi- tionally, glutathione peroxidase was decreased in hippocampus, and thiobarbaturic acid-reactive species (TBARS; indicative of lipid peroxi- dation) were markedly elevated in liver and cerebral cortex of aldh5a1−/− mice. The data of Table 2, highlighting decreased levels of mRNA encoding catalase, superoxide dismutase, glutathione peroxidase and glutathione transferase in untreated aldh5a1−/− mice, are consis- tent with the previously published enzyme studies in tissues, and have been further corroborated in the current study using Western blot analyses (Fig. 5). Further, Table 2 revealed alterations in the mRNA levels in aldh5a1−/− mice that encode peroxiredoxins, a family of antioxidant enzymes that also control cytokine-induced peroxide levels and thereby mediate signal transduction in mammalian cells [52]. Additionally, we observed a significant decrease in the level of mRNA encoding aldh1a1 in the liver of aldh5a1−/− mice that was returned to levels that were not significantly different from aldh5a1+/+ mice using Tor 2 (Table 2).
5.Conclusions
We provide metabolic, protein and molecular genetic data supporting the concept that inhibition of mTOR has therapeutic rele- vance in aldh5a1−/− mice, and by extension, patients with SSADHD.The Transparency Torin 2 document associated with this article can be found in the online version.