Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy

Abstract

Purpose: Adenosine kinase (ADK) represents the key metabolic enzyme for the regulation of extracellular adenosine levels in the brain. In adult brain, ADK is primarily present in astrocytes. Several lines of experimental evidence support a critical role of ADK in different types of brain injury associated with astrogliosis, which is also a prominent morphologic feature of temporal lobe epilepsy (TLE). We hypothesized that dysregulation of ADK is an ubiquitous pathologic hallmark of TLE.

Methods: Using immunocytochemistry and Western blot analysis, we investigated ADK protein expression in a rat model of TLE during epileptogenesis and the chronic epileptic phase and compared those findings with tissue resected from TLE patients with mesial temporal sclerosis (MTS).

Key findings: In rat control hippocampus and cortex, a low baseline expression of ADK was found with mainly nuclear localization. One week after the electrical induction of status epilepticus (SE), prominent up-regulation of ADK became evident in astrocytes with a characteristic cytoplasmic localization. This increase in ADK persisted at least for 3-4 months after SE in rats developing a progressive form of epilepsy. In line with the findings from the rat model, expression of astrocytic ADK was also found to be increased in the hippocampus and temporal cortex of patients with TLE. In addition, in vitro experiments in human astrocyte cultures showed that ADK expression was increased by several proinflammatory molecules (interleukin-1β and lipopolysaccharide).

Significance: These results suggest that dysregulation of ADK in astrocytes is a common pathologic hallmark of TLE. Moreover, in vitro data suggest the existence of an additional layer of modulatory crosstalk between the astrocyte-based adenosine cycle and inflammation. Whether this interaction also can play a role in vivo needs to be further investigated.

Figure 1. Immunostaining of ADK in hippocampal tissue of control rat and after induction of status epilepticus (SE)

Panels A-B: Control hippocampus showing low ADK immunoreactivity (IR) in the different hippocampal subfields; B: CA3 region showing no detectable IR in neurons and sparse nuclear IR in glial cells (inset). Panels C-H: hippocampus 1 week post-SE showing increased ADK expression in glial cells within the different hippocampal subfields, including CA3 (D), CA1 (E-F; arrows), DG (G-H, arrows; arrowheads in G indicate ADK positive cells in the subgranular zone) regions; inset a in D shows ADK positive astrocytes in CA3; inset b in D: merged confocal image, showing ADK positive glial cells (red) surrounding the NeuN (green) positive neuronal cells in CA3. Inset C in D: ADK (red) NeuN (green) co-localization. Panel E: merged confocal image, showing ADK positive glial cells (red) surrounding the NeuN (green) in CA1; inset in G: absence of co-localization of ADK (red) and DCX (green) in the subgranular zone; inset in H shows expression of ADK (red) in astrocytes (GFAP positive, green). Scale bars: A, C: 560 μm; B, D: 140 μm. E: 20 μm; G: 70 μm; F, H: 40 μm.

Figure 2. ADK immunoreactivity (IR) in rat hippocampus 3-4 month after SE

Panels A, C, E, G: hippocampus of rats with long term epilepsy, progressive (pLT; ~10 seizures/day; last seizure less than 2 hours before sacrifice) showing both nuclear and cytoplasmic ADK IR in different hippocampal subfields, including CA3 (C; inset), CA1 (E; inset a) and dentate gyrus (DG, G; inset); Inset in C shows ADK positive glial cells surrounding a neuron with weak ADK. inset b in E shows expression of ADK (red) in astrocytes (GFAP positive, green). Panels B, D, F, H: hippocampus of rats with long term epilepsy, non-progressive (npLT; 1 seizure every other day; last seizure at least 1 day before sacrifice), showing low ADK IR in different hippocampal subfields; mainly nuclear IR is observed in sparse glial cells in CA3 (D, inset), CA1 (F) and DG (H; inset). Scale bars: A, C: 280 μm; C, D: 140 μm. E-H: 70 μm.

Figure 3. Evaluation of ADK astroglial immunoreactivity in hippocampal regions of control rat and after induction of status epilepticus (SE)

Bar diagrams of ADK-positive cells in CA1 (A), CA3 (B) and dentate gyrus (hilar region; C) of control hippocampus and hippocampus at 24 h, 1 week and 3-4 months post-SE (rats with long term epilepsy, progressive, pLT (~10 seizures/day; last seizure less than 2 hours before sacrifice) and rats with long term epilepsy, non-progressive, npLT (1 seizure every other day; last seizure at least 1 day before sacrifice). Nuclear and extranuclear staining: *p< 0.05 vs control, one-way ANOVA followed by Tukey's test.

Figure 4. Western blot analysis of ADK in temporal cortex of control rat and after induction of status epilepticus (SE)

Panel A: representative immunoblots of total homogenates from control rat cortex and after induction of SE (24 h, 1 week, 3-4 months long term progressive epilepsy, pLT, and non-progressive, n-pLT). Panel B: densitometric analysis: values (optical density units, O.D.) are mean ± SEM , (control, n=6; 24 h, n=4; 1 week, n=6; pLT, n=5; npLT, n=4) , relative to the optical density of β-actin; *, p < 0.05 compared to controls.

Figure 5. Distribution of ADK immunoreactivity in the hippocampus and temporal cortex (Ctx) of control and TLE patients with mesial temporal sclerosis (MTS)

Panels A, C, E: control hippocampus (A, CA3; C, dentate gyrus, hilar region) and Ctx (E) showing weak ADK immunoreactivity (IR) in both glial and neuronal cells (insets a, b in E). Sections are counterstained with hematoxylin. Histologically normal surgical hippocampus and cortex displayed a pattern of IR similar to that observed in control autopsy hippocampus (not shown). Panels B and F: MTS, hippocampal sclerosis (B, CA3; F, dentate gyrus, hilar region), showing increased ADK expression. Expression was observed in residual neurons (arrowhead in D) and in reactive astrocytes (arrows in D). Panel F: MTS, temporal cortex showing increased ADK expression with both neuronal and glial IR (insets). Scale bars: A, B: 160 μm; C, D 40 μm; E, F 40 μm. Western blot analysis of ADK in temporal cortex: representative immunoblots (G) and densitometric analysis (H) of total homogenates from autopsy control temporal cortex and surgical cortex from MTS and non-MTS specimens. H: densitometric analysis: values (optical density units, O.D.) are mean ± SEM, 5 controls, 5 MTS and 4 non-MTS, relative to the optical density of β-actin; * P< 0.05.

Figure 6. ADK in human astrocytes induced by IL-1β and LPS

Panel A: Representative immunoblot of total homogenates from human fetal astrocytes untreated and treated for 24 h with 10 ng/ml IL-1β or 100 ng/ml LPS. Panel B: Densitometric analysis: values (optical density units, O.D.) are mean ± SEM, (control, n=3; IL-1β, n=4 and LPS, n=4 treated astrocytes), relative to the optical density of β-actin; *, p < 0.05, compared to controls.