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. 2018 Nov:71:241-254.
doi: 10.1016/j.neurobiolaging.2018.08.002. Epub 2018 Aug 7.

Altered glutamate clearance in ascorbate deficient mice increases seizure susceptibility and contributes to cognitive impairment in APP/PSEN1 mice

Deborah J Mi  1 , Shilpy Dixit  1 , Timothy A Warner  2 , John A Kennard  1 , Daniel A Scharf  3 , Eric S Kessler  3 , Lisa M Moore  3 , David C Consoli  4 , Corey W Bown  4 , Angeline J Eugene  3 , Jing-Qiong Kang  2 , Fiona E Harrison  5
Affiliations

Altered glutamate clearance in ascorbate deficient mice increases seizure susceptibility and contributes to cognitive impairment in APP/PSEN1 mice

Deborah J Mi et al. Neurobiol Aging. 2018 Nov.

Abstract

Ascorbate (vitamin C) is critical as a first line of defense antioxidant within the brain, and specifically within the synapse. Ascorbate is released by astrocytes during glutamate clearance and disruption of this exchange mechanism may be critical in mediating glutamate toxicity within the synapse. This is likely even more critical in neurodegenerative disorders with associated excitotoxicity and seizures, in particular Alzheimer's disease, in which ascorbate levels are often low. Using Gulo-/- mice that are dependent on dietary ascorbate, we established that low brain ascorbate increased sensitivity to kainic acid as measured via behavioral observations, electroencephalography (EEG) measurements, and altered regulation of several glutamatergic system genes. Kainic acid-induced immobility was improved in wild-type mice following treatment with ceftriaxone, which upregulates glutamate transporter GLT-1. The same effect was not observed in ascorbate-deficient mice in which sufficient ascorbate is not available for release. A single, mild seizure event was sufficient to disrupt performance in the water maze in low-ascorbate mice and in APPSWE/PSEN1dE9 mice. Together, the data support the critical role of brain ascorbate in maintaining protection during glutamatergic hyperexcitation events, including seizures. The study further supports a role for mild, subclinical seizures in cognitive decline in Alzheimer's disease.

Keywords: Ascorbate; Behavior; GLT-1; Glutamate; Seizure; Vitamin C.

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Conflict of interest statement

Conflicts of Interest. The authors have no competing conflicts of interest to report.

Figures

Figure 1.
Figure 1.. Glutamate uptake-ascorbate release exchange mechanism in astrocytes.
The two tethered systems (red box) of glutamate uptake via GLT-1, and ascorbate release through volume regulated anion channels (VRAC) are highlighted. Under normal conditions, as glutamate enters the astrocyte, it causes cellular swelling. This change results in the opening of volume regulated anion channels (VRACs), which allow ascorbate (ASC, yellow circles) to efflux from the astrocyte into the synapse. As an antioxidant, ascorbate donates electrons as needed to radical species in the synaptic cleft, eventually becoming oxidized to dehydroascorbic acid within the synapse (DHA, brown circles). Alternatively, some ascorbate is also available for uptake by neurons on the Sodium Dependent Vitamin C Transporter, type 2 (SVCT2). Dehydroascorbic acid is taken up on glucose transporters (GLUTS) where it is reduced back to ascorbate, ready for release owing to its very efficient recycling chemistry (Harrison and May, 2009,Wilson, 1997,Wilson, et al., 2000). Glutamate (dark green circles) is converted to glutamine (light green circles), which is released from the astrocyte for reuptake by neurons. Slower glutamate clearance can contribute to hyper-stimulation of post-synaptic receptors, and contribute to localized oxidative stress, with the potential to further damage GLT-1 function.
Figure 2.
Figure 2.. Increased seizure susceptibility to kainic acid but not pilocarpine or PTZ in low ascorbate-supplemented Gulo−/− mice.
Gulo−/− mice were scored according to severity of behavioral response following treatment with (A) kainic acid (10 mg/kg) N=19 high ascorbate (1.0 g/L), N=20 low ascorbate (0.03 g/L), (B) pilocarpine (40 mg/kg) N=6 high ascorbate, N=9 low ascorbate, or (C) PTZ (40 mg/kg) N=20 high ascorbate, N=17 low ascorbate. Primary output measure for kainic acid and pilocarpine was latency to onset of Stage 3 of the Racine scale (head bob, and/or other repetitive behavior), as well as noting any overt seizure occurrences corresponding to Stages 4–6 of the Racine scale. For PTZ the number of small and large full body tics were scored. (D) Brain ascorbate was measured in cerebellum N=26 High, N=15 Low. Data analyzed by unpaired t-test with Welch’s correction where variances differed significantly between groups. *** P<0.001 different from high ascorbate condition. ASC, Ascorbate.
Figure 3.
Figure 3.. Greater abnormal EEG and seizures activities in low ascorbate Gulo−/− mice at baseline and following kainic acid.
Gulo−/− mice on high (1.0 g/L, N=10) and low (0.03 g/L, N=8) ascorbate (ASC) supplementation were monitored via skull-mounted EEG devices for 24 hours prior to-and 60 mins. following treatment with 10 mg/kg kainic acid. At baseline more (A) spike wave discharges, and (B) myoclonic jerks were observed in mice on low ascorbate treatments. Following kainic acid low ascorbate mice experienced more (C) spike discharges, (D) myoclonic jerks, (E) tonic-clonic seizures, and (F) tonic clonic-like seizures than control, high-ascorbate mice. (G-I) Representative EEG recordings show (G) slow spike-wave discharges (SWDs), (H) myoclonic jerks and (I) generalized tonic clonic seizures from the Gulo−/− mice supplemented with low ascorbate (ASC). Unpaired t-tests, * P<0.05, ** P<0.01 from high ascorbate controls.
Figure 4.
Figure 4.. Ceftriaxone upregulates GLT-1 but does not protect against kainic acid-induced behavioral changes.
A) Wild-type mice pre-treated with 200 mg/kg ceftriaxone daily for 14 days spent less time immobile in the 30 mins following treatment with 10 mg/kg kainic acid. Saline N=11, ceftriaxone N=9. B-C) Illustration of activity data output from FPA chambers showing B) spikes representative of myoclonic jerks (red arrows) and C) Movement in the chambers in the initial few minutes of testing following kainic acid administration. D-F) High (1.0 g/L) and low (0.03 g/L) ascorbate treated Gulo−/− mice pre-treated with 14 days CFX (200 mg/kg) did not differ in three automated or semi-automated measures of activity in the Force Plate Actimeters D) Bouts of low mobility, E) Spikes reflecting possible myoclonic jerks, and F) Distance travelled. (High SAL N=9, High ceftriaxone n=11, Low SAL N=11, Low ceftriaxone N=13). G,I) GLT-1 expression was significantly increased in low ascorbate Gulo−/− mice with a further increase low ascorbate mice according to ceftriaxone treatment (N=7–9 per group, from 4 separate blots). H,I) xCT expression was increased in low ascorbate Gulo−/− mice but did not changes in response to ceftriaxone (N=4–5 per group). J) Brain ascorbate levels reflected dietary supplementation regimens. * P<0.05, ***P<0.001 differences between groups as marked.
Figure 5.
Figure 5.. Single kainic acid-induced seizures can impact spatial learning and memory in young mice.
(A) Increased locomotor activity levels in APP/PSEN1 and SVCT2+/−APP/PSEN1 mice. The four genotypes did not differ on acquisition of the water maze task for either (B) Visible or (C) Hidden platform acquisition. (D) Shorter latencies to show head bob behaviors (Stage 3 of Racine scale) following kainic acid (10 mg/kg) treatment indicated greater sensitivity in all three mutant genotypes compared to wild-type mice. One mouse died following seizure initiation and so is included in cued, and hidden platform acquisition data only. Time spent in target “T” versus non-target quadrants (Left “L”, Opposite “O” and Right “R”) during (E) first, and ( F) second probe trials. (G) Comparison of Search Error, average distance from the platform during probe trials performed pre-and post-kainic acid injection indicated that all mice performed more poorly on the second test, and this difference was significant in wild-type, SVCT2+/− and SVCT2+/−APP/PSEN1 mice. (F) Reversal learning of a new hidden platform position following kainic acid was impaired in APP/PSEN1 mice on days 1 and 2. (G) There was a significant correlation between seizure Stage 3 onset latency and recall of previously learned platform position for SVCT2+/−APP/PSEN1 mice only, dotted line. *, *** P<0.05, P<0.001 genotype different from WT; a P<0.05, b P<0.01, c P<0.001 from chance performance (dashed line); δP<0.05, Probe 2 different from Probe one within genotype. Wild-type (WT) N=17, SVCT2+/−N=9, APP/PSEN1 N=8, SVCT2+/−APP/PSEN1 N=9.
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