Astrogliosis is a pathological hallmark of the epileptic brain. The identification of mechanisms that link astrogliosis to neuronal dysfunction in epilepsy may provide new avenues for therapeutic intervention. Here we show that astrocyte-expressed adenosine kinase (ADK), a key negative regulator of the brain inhibitory molecule adenosine, is a potential predictor and modulator of epileptogenesis. In a mouse model of focal epileptogenesis, in which astrogliosis is restricted to the CA3 region of the hippocampus, we demonstrate that upregulation of ADK and spontaneous focal electroencephalographic seizures were both restricted to the affected CA3. Furthermore, spontaneous seizures in CA3 were mimicked in transgenic mice by overexpression of ADK in this brain region, implying that overexpression of ADK without astrogliosis is sufficient to cause seizures. Conversely, after pharmacological induction of an otherwise epileptogenesis-precipitating acute brain injury, transgenic mice with reduced forebrain ADK were resistant to subsequent epileptogenesis. Likewise, ADK-deficient ES cell-derived brain implants suppressed astrogliosis, upregulation of ADK, and spontaneous seizures in WT mice when implanted after the epileptogenesis-precipitating brain injury. Our findings suggest that astrocyte-based ADK provides a critical link between astrogliosis and neuronal dysfunction in epilepsy.

A ketogenic diet (KD) is a high-fat, low-carbohydrate metabolic regimen; its effectiveness in the treatment of refractory epilepsy suggests that the mechanisms underlying its anticonvulsive effects differ from those targeted by conventional antiepileptic drugs. Recently, KD and analogous metabolic strategies have shown therapeutic promise in other neurologic disorders, such as reducing brain injury, pain, and inflammation. Here, we have shown that KD can reduce seizures in mice by increasing activation of adenosine A 1 receptors (A 1 Rs). When transgenic mice with spontaneous seizures caused by deficiency in adenosine metabolism or signaling were fed KD, seizures were nearly abolished if mice had intact A 1 Rs, were reduced if mice expressed reduced A 1 Rs, and were unaltered if mice lacked A 1 Rs. Seizures were restored by injecting either glucose (metabolic reversal) or an A 1 R antagonist (pharmacologic reversal). Western blot analysis demonstrated that the KD reduced adenosine kinase, the major adenosine-metabolizing enzyme. Importantly, hippocampal tissue resected from patients with medically intractable epilepsy demonstrated increased adenosine kinase. We therefore conclude that adenosine deficiency may be relevant to human epilepsy and that KD can reduce seizures by increasing A 1 R-mediated inhibition.

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Background Breast cancer angiogenesis is key for metastasis and predicts a poor prognosis. Angiotensin-converting enzyme 2 (ACE2), as a member of the renin-angiotensin system (RAS), was reported to restrain the progression of hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC) through inhibiting angiogenesis. However, the relationship between ACE2 and breast cancer angiogenesis remains unclear. Methods The prognosis and relative gene selection were analysed using the GEPIA, GEO, TCGA and STRING databases. ACE2 expression in breast cancer tissue was estimated by reverse transcription-quantitative polymerase chain reaction (qPCR). Breast cancer cell migration, proliferation and angiogenesis were assessed by Transwell migration, proliferation, tube formation, and wound healing assays. The expression of vascular endothelial growth factor A (VEGFa) was detected by qPCR and Western blotting. The phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), mitogen-activated protein kinase 1/2 (MEK1/2), and extracellular signal-regulated protein kinase 1/2 (ERK1/2) was examined by Western blotting. Breast cancer metastasis and angiogenesis in vivo were measured using a zebrafish model. Results ACE2 was downregulated in breast cancer patients. Patients with higher ACE2 expression had longer relapse-free survival (RFS). In vitro, ACE2 inhibited breast cancer migration. Meanwhile, ACE2 in breast cancer cells inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, tube formation and migration. In the zebrafish model, ACE2 inhibited breast cancer cell metastasis, as demonstrated by analyses of the number of disseminated foci and the metastatic distance. Neo-angiogenesis was also decreased by ACE2. ACE2 downregulated the expression of VEGFa in breast cancer cells. Furthermore, ACE2 in breast cancer cells inactivated the phosphorylation of VEGFR2, MEK1/2, and ERK1/2 in HUVECs. Conclusions Our findings suggest that ACE2, as a potential resister to breast cancer, might inhibit breast cancer angiogenesis through the VEGFa/VEGFR2/ERK pathway. Trial registration Retrospectively registered. Electronic supplementary material The online version of this article (10.1186/s13046-019-1156-5) contains supplementary material, which is available to authorized users.

Epilepsy is characterized by both neuronal and astroglial dysfunction. The endogenous anticonvulsant adenosine, the level of which is largely controlled by astrocytes, might provide a critical link between astrocyte and neuron dysfunction in epilepsy. Here we studied astrogliosis, a hallmark of the epileptic brain, adenosine dysfunction and the emergence of spontaneous seizures in a comprehensive approach including a new mouse model of focal epileptogenesis, mutant mice with altered brain levels of adenosine, and mice lacking adenosine A 1 receptors. In wild type mice, following a focal epileptogenesis-precipitating injury, astrogliosis, upregulation of the adenosineremoving astrocytic enzyme adenosine kinase (ADK), and spontaneous seizures all coincided in a spatio-temporally restricted manner. Importantly, these spontaneous seizures were mimicked in untreated transgenic mice overexpressing ADK in brain or those lacking A 1 receptors. Conversely, mice with reduced forebrain ADK did not develop astrogliosis or spontaneous seizures. Our studies define ADK as critical upstream regulator of A 1 receptor mediated modulation of neuronal excitability and support the ADK hypothesis of epileptogenesis implicating that upregulation of ADK during astrogliosis provides a critical link between astrocyte and neuron dysfunction in epilepsy. These findings define ADK as rational target for therapeutic intervention.

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Adenosine augmentation therapies (AAT) make rational use of the brain's own adenosine-based seizure control system and hold promise for the therapy of refractory epilepsy. In an effort to develop an AAT compatible with future clinical application, we developed a novel silk protein-based release system for adenosine. Adenosine releasing brain implants with target release doses of 0, 40, 200, and 1,000 ng adenosine/day were prepared by embedding adenosine-containing microspheres into nanofilm-coated silk fibroin scaffolds. In vitro, the respective polymers released 0, 33.4, 170.5, and 819.0 ng adenosine/day over 14 days. The therapeutic potential of the implants was validated in a dose-response study in the rat model of kindling epileptogenesis. Four days prior to the onset of kindling, adenosine-releasing polymers were implanted into the infrahippocampal cleft and progressive acquisition of kindled seizures was monitored over a total of 48 stimulations. We document a dose-dependent retardation of seizure acquisition. In recipients of polymers releasing 819 ng adenosine/day, kindling epileptogenesis was delayed by one week corresponding to 18 kindling stimulations. Histological analysis of brain samples confirmed correct location of implants and electrodes. We conclude that silk-based delivery of around 1,000 ng adenosine/day is a safe and efficient strategy to suppress seizures.