Adenosine Kinase Deficiency in the Brain Results in Maladaptive Synaptic Plasticity

Abstract

Adenosine kinase (ADK) deficiency in human patients (OMIM:614300) disrupts the methionine cycle and triggers hypermethioninemia, hepatic encephalopathy, cognitive impairment, and seizures. To identify whether this neurological phenotype is intrinsically based on ADK deficiency in the brain or if it is secondary to liver dysfunction, we generated a mouse model with a brain-wide deletion of ADK by introducing a Nestin-Cre transgene into a line of conditional ADK deficient Adk^fl/fl mice. These Adk^Δbrain mice developed a progressive stress-induced seizure phenotype associated with spontaneous convulsive seizures and profound deficits in hippocampus-dependent learning and memory. Pharmacological, biochemical, and electrophysiological studies suggest enhanced adenosine levels around synapses resulting in an enhanced adenosine A₁ receptor (A₁R)-dependent protective tone despite lower expression levels of the receptor. Theta-burst-induced LTP was enhanced in the mutants and this was dependent on adenosine A_2A receptor (A_2AR) and tropomyosin-related kinase B signaling, suggesting increased activation of these receptors in synaptic plasticity phenomena. Accordingly, reducing adenosine A_2A receptor activity in Adk^Δbrain mice restored normal associative learning and contextual memory and attenuated seizure risk. We conclude that ADK deficiency in the brain triggers neuronal adaptation processes that lead to dysregulated synaptic plasticity, cognitive deficits, and increased seizure risk. Therefore, ADK mutations have an intrinsic effect on brain physiology and may present a genetic risk factor for the development of seizures and learning impairments. Furthermore, our data show that blocking A_2AR activity therapeutically can attenuate neurological symptoms in ADK deficiency.

Significance statement: A novel human genetic condition (OMIM #614300) that is based on mutations in the adenosine kinase (Adk) gene has been discovered recently. Affected patients develop hepatic encephalopathy, seizures, and severe cognitive impairment. To model and understand the neurological phenotype of the human mutation, we generated a new conditional knock-out mouse with a brain-specific deletion of Adk (Adk^Δbrain). Similar to ADK-deficient patients, Adk^Δbrain mice develop seizures and cognitive deficits. We identified increased basal synaptic transmission and enhanced adenosine A_2A receptor (A_2AR)-dependent synaptic plasticity as the underlying mechanisms that govern these phenotypes. Our data show that neurological phenotypes in ADK-deficient patients are intrinsic to ADK deficiency in the brain and that blocking A_2AR activity therapeutically can attenuate neurological symptoms in ADK deficiency.

Keywords: adenosine kinase; epilepsy; gene mutation; human genetic disorder; learning and memory; mouse model.

Figure 1.

Brain-specific deletion of ADK in Adk^Δbrain mice. A, Transgenic strategy: exon 7 of the Adk allele is flanked with loxP sites in Adk^fl/fl mice. B, Adk PCR on genomic DNA extracts from the cortex (C), striatum (S), hippocampus (Hp), cerebellum (Cb), heart (Ht), lung (L), liver (Lv), and spleen (Sp) from Adk^fl/fl and Adk^Δbrain mice. Tail DNA from an Adk^fl/fl (T) and wild-type mouse (WT) were included as positive controls. Water (−) was included as a no-template control. Adk forward and reverse primer (P1 and P2) sites are depicted in A. C, ADK (40 kDa) Western blots on cortical, striatal, and hippocampal protein extracts from Adk^fl/fl and Adk^Δbrain mice; n = 2/genotype are used as representatives. ADK Western blots were reprobed with GAPDH (37 kDa) as a loading control. D, E, ADK immunohistochemistry of cortical brain tissue from Adk^fl/fl (D) and Adk^Δbrain (E) mice. F, G, Nissl stain of hippocampal formation from Adk^fl/fl (F) and Adk^Δbrain (G) mice.

Figure 2.

Loss of brain ADK results in increased susceptibility to seizure induction. A, Representative cortical EEG trace that includes a complete DPCPX- induced seizure (110 s) in an Adk^Δbrain mouse that received a total cumulative dose of 2.0 mg/kg DPCPX intraperitoneally. High-resolution portions (15 s) of the DPCPX-induced seizure are depicted in cortical/hippocampal EEG¹ and EEG². These EEG traces correspond to boxes demarcated with 1 and 2 in upper cortical trace. See Movie 2 for a matching behavioral seizure. B, DPCPX does not trigger seizures in Adk^fl/fl control mice. Shown is a representative section of a cortical EEG trace (110 s) recorded after a cumulative dose of 3.5 mg/kg DPCPX intraperitoneally. C, DPCPX dose response (0.5 mg/kg, i.p., every 10 min) in Adk^Δbrain mice (n = 6) and Adk^fl/fl mice (n = 5). D, E, DPCPX (3 mg/kg, i.p.) administered to Adk^Δbrain mice (n = 10) causes status epilepticus (D, ****p < 0.0001) and increased mortality (E, ****p < 0.0001) compared with DPCPX-treated Adk^fl/fl mice (n = 10), vehicle-treated Adk^fl/fl mice (n = 6), and vehicle-treated Adk^Δbrain mice (n = 6). Statistical analysis: log–rank (Mantel–Cox) test.

Figure 3.

Loss of brain ADK results in spontaneous seizures. A, Thirty seconds of representative hippocampal EEG traces from Adk^Δbrain mice and C57BL6 (WT), Nestin-Cre^+/−:Adk^+/+ (Nes-Cre^+/−), and Adk^fl/fl control mice demonstrate comparable baseline seizure activity. B, Representative cortical EEG recording of a complete spontaneous convulsive seizure (55 s) from an Adk^Δbrain mouse (top trace). Bottom traces are high-resolution cortical and hippocampal EEG recordings of a 20 s portion of seizure demarcated by the box in top trace. See Movie 3 for a matching behavioral seizure.

Figure 4.

ADK deficiency in the brain results in cognitive impairment. A, Associative learning in the conditioned freezing paradigm is significantly decreased during conditioned stimulus 2 (CS2) in Adk^Δbrain (n = 13) mice, compared with WT (n = 15), Nestin-Cre^+/− (n = 10), and Adk^fl/fl (n = 14) controls. Within Adk^Δbrain mice, the percentage time freezing during CS3 and CS4 is significantly increased compared with baseline CS1 freezing. B, Contextual freezing is significantly impaired in Adk^Δbrain mice (n = 13), compared with WT (n = 15), Nestin-Cre^+/− (n = 10), and Adk^fl/fl (n = 14) controls. Data are represented as the mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ##p < 0.01.

Figure 5.

Adk^Δbrain mice have increased synaptic adenosine. A, Disinhibition of synaptic transmission by the A₁R antagonist DPCPX (50 nm) is facilitated in Adk^Δbrain vs Adk^fl/fl slices (n = 5 mice each). DPCPX increases the fEPSP slope in Adk^Δbrain slices versus baseline. B, Percentage change in fEPSP slope during the last 10 min of DPCPX. C, Decreased A₁R mRNA levels of Adk^Δbrain (n = 6) versus Adk^fl/fl (n = 4 mice) cortex. D, Binding curve for [³H]DPCPX is reduced the Adk^Δbrain versus Adk^fl/fl cortex (n = 4 mice each). E, Average B_max values from the binding curves of Adk^Δbrain versus Adk^fl/fl mice (B_max: 73 ± 6.5 vs 239 ± 13.4 fmol/mg protein). Data are presented as the mean ± SEM. 100% corresponds to the averaged fEPSP recorded 10 min before drug perfusion (A, B, F, G). F, Input–output curves were obtained from hippocampal slices to address changes in synaptic transmission level. The input–output curves correspond to responses generated by increasing stimulation intensities (60–340 mA) in Adk^fl/fl (n = 5) and Adk^Δbrain (n = 5) mice. Results are shown as the mean ± SEM and statistical analysis was performed using an F test (*p < 0.05). *p < 0.05, **p < 0.01, ***p < 0.001, ##p < 0.01, ####p < 0.0001.

Figure 6.

Adk^Δbrain mice show enhanced A_2A and TrkB receptor- dependent LTP. A, B, The A_2AR-selective agonist CGS21680 (10 nm) and antagonist SCH58261 (50 nm) does not affect the fEPSP slope in Adk^fl/fl and Adk^Δbrain slices (n = 4 mice/genotype/drug). C–E, SCH58261 restores normal theta-burst (3 × 3)-induced LTP in Adk^Δbrain mice. C, D, Average change in fEPSP slopes in the absence versus presence of SCH58261 in Adk^fl/fl (C) and Adk^Δbrain (D) slices (n = 5 mice/genotype/drug). E, Percentage of change in the fEPSP slopes recorded at 52–60 min after LTP induction. F, The TrkB inhibitor K252a (200 nm) restores normal theta-burst (3 × 3)-induced LTP in Adk^Δbrain slices (absence: 150 ± 6.7% vs presence: 101 ± 7.0%, n = 5 mice each,). Insets in C, D, and F are representative traces of six consecutive responses composed of the stimulus artifact, presynaptic volley, and fEPSP obtained before (1, 3) and after (2, 4) the theta-burst stimuli in the absence (1, 2) and presence (3, 4) of drug. LTP was significant (####) after theta-burst stimuli versus baseline (C, D, F). G, Representative blot of full-length TrkB receptor (FL-TrkB, 145 kDa), truncated TrkB receptor (TrkB-Tc, 95 kDa), and β-tubulin (55 kDa, loading control) is shown. H, Averaged immunodensities of TrkB receptor normalized to β-tubulin and the ratio of FL-TrkB to TrkB-Tc (n = 8 mice/genotype). Results are presented as mean ± SEM. 100% corresponds to the averaged fEPSP slope recorded 10 min before LTP induction. *p < 0.05, **p < 0.01, ####p < 0.0001.

Figure 7.

Blockade of A_2AR activity ameliorates the inducible seizure phenotype and cognitive impairment in Adk^Δbrain mice. A, Increased survival time of Adk^Δbrain mice treated with SCH 58261 (0.5 mg/kg, i.p., 30 min in advance, n = 6) or Ana-12 (0.5 mg/kg, i.p., 4 h in advance, n = 6) before the DPCPX (3 mg/kg, i.p.) challenge compared with DPCPX controls (n = 10). B, Contingency analysis indicates a significant decrease in the percentage of Adk^Δbrain:A_2AR^−/− mice (3 of 10) that develop the inducible seizure phenotype compared with Adk^Δbrain (12 of 12) and Adk^Δbrain:A₁R^−/− (8 of 8) mice. C, Kaplan–Meier survival curve indicating increased Adk^Δbrain:A₁R^−/− (n = 18) mortality compared with Adk^Δbrain (n = 18) and Adk^Δbrain:A_2AR^−/− (n = 10). D, Associative learning indexed as total percentage time freezing during the CS 2–4 is restored to Adk^fl/fl (n = 13) control levels in Adk^Δbrain:A_2AR^−/− (n = 12) mice and is significantly increased compared with Adk^Δbrain (n = 13) mice. E, Contextual memory indexed as total percentage time freezing during the 8 min contextual freezing trial is significantly increased in Adk^Δbrain:A_2AR^−/− (n = 12) and Adk^fl/fl (n = 13) mice, compared with Adk^Δbrain (n = 13) mice. Data are represented as the mean ± SEM; **p < 0.01, ***p < 0.001, ****p < 0.0001.