Theresa A. Lusardi

Adenosine kinase is a target for the prediction and prevention of epileptogenesis in mice.

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.

Ischemic preconditioning regulates expression of microRNAs and a predicted target, MeCP2, in mouse cortex

Preconditioning describes the ischemic stimulus that triggers an endogenous, neuroprotective response that protects the brain during a subsequent severe ischemic injury, a phenomenon known as ‘tolerance’. Ischemic tolerance requires new protein synthesis, leads to genomic reprogramming of the brains response to subsequent ischemia, and is transient. MicroRNAs (miRNAs) regulate posttranscriptional gene expression by exerting direct effects on messenger RNA (mRNA) translation. We examined miRNA expression in mouse cortex in response to preconditioning, ischemic injury, and tolerance. The results of our microarray analysis revealed that miRNA expression is consistently altered within each group, but that preconditioning was the foremost regulator of miRNAs. Our bioinformatic analysis results predicted that preconditioning-regulated miRNAs most prominently target mRNAs that encode transcriptional regulators; methyl-CpG binding protein 2 (MeCP2) was the most prominent target. No studies have linked MeCP2 to preconditioning or tolerance, yet miR-132, which regulates MeCP2 expression, is decreased in preconditioned cortex. Downregulation of miR-132 is consistent with our finding that preconditioning ischemia induces a rapid increase in MeCP2 protein, but not mRNA, in mouse cortex. These studies reveal that ischemic preconditioning regulates expression of miRNAs and their predicted targets in mouse brain cortex, and further suggest that miRNAs and MeCP2 could serve as effectors of ischemic preconditioning-induced tolerance.

Epigenetic changes induced by adenosine augmentation therapy prevent epileptogenesis

Epigenetic modifications, including changes in DNA methylation, lead to altered gene expression and thus may underlie epileptogenesis via induction of permanent changes in neuronal excitability. Therapies that could inhibit or reverse these changes may be highly effective in halting disease progression. Here we identify an epigenetic function of the brains endogenous anticonvulsant adenosine, showing that this compound induces hypomethylation of DNA via biochemical interference with the transmethylation pathway. We show that inhibition of DNA methylation inhibited epileptogenesis in multiple seizure models. Using a rat model of temporal lobe epilepsy, we identified an increase in hippocampal DNA methylation, which correlates with increased DNA methyltransferase activity, disruption of adenosine homeostasis, and spontaneous recurrent seizures. Finally, we used bioengineered silk implants to deliver a defined dose of adenosine over 10 days to the brains of epileptic rats. This transient therapeutic intervention reversed the DNA hypermethylation seen in the epileptic brain, inhibited sprouting of mossy fibers in the hippocampus, and prevented the progression of epilepsy for at least 3 months. These data demonstrate that pathological changes in DNA methylation homeostasis may underlie epileptogenesis and reversal of these epigenetic changes with adenosine augmentation therapy may halt disease progression.

Effect of acute calcium influx after mechanical stretch injury in vitro on the viability of hippocampal neurons

We use a new in vitro model to examine the effect of mechanical deformation on neurons. We examined acute changes in cytosolic calcium concentrations ([Ca(2+)](i)) caused by a rapid stretch of cultured hippocampal neurons, using mechanical loading conditions that mimic brain deformations during trauma. We found that stretch-injury of neurons induces a strain-dependent increase in [Ca(2+)](i). Remarkably, the extent of this calcium response exceeded the levels initiated by chemical toxicity with NMDA (100 microM) or glutamate (5 mM) exposure. Propidium iodide labeling at 24 h following stretch showed neuronal death occurred only at the most severe level of mechanical injury. Although NMDA-induced toxicity could be inhibited in calcium free media or by treatment with MK-801, stretch-induced neuronal death was not similarly reduced with either treatment. Unexpectedly, reduction of the acute stretch-induced calcium transient with calcium-free media or MK-801 resulted in an increase in neuronal death at lower stretch levels. These data suggest that mechanical stretch can initiate calcium influx in hippocampal neurons, but substantially modulating the early calcium flux from the extracellular space or through the NMDA channel does not provide an effective means for improving neuronal survival.

Ubiquitin proteasome-mediated synaptic reorganization: a novel mechanism underlying rapid ischemic tolerance.

Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (gene-mediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin–proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132), MG115 (carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal), and clasto-lactacystin-β-lactone]. A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin–proteasome system, rendering neurons resistant to excitotoxicity.

Antiepileptic effects of silk-polymer based adenosine release in kindled rats.

Pharmacotherapy for epilepsy is limited by high incidence of pharmacoresistance and failure to prevent development and progression of epilepsy. Using the rat hippocampal kindling model, we report on the therapeutic potential of novel silk-based polymers engineered to release the anticonvulsant adenosine. Polymers were designed to release 1000 ng adenosine per day during a time span of ten days. In the first experiment rats were kindled by hippocampal electrical stimulation until all animals reacted with stage 5 seizures. Adenosine-releasing or control polymers were then implanted into the infrahippocampal fissure ipsilateral to the site of stimulation. Subsequently, only recipients of adenosine-releasing implants were completely protected from generalized seizures over a period of ten days corresponding to the duration of sustained adenosine release. To monitor seizure development in the presence of adenosine, adenosine-releasing or control polymers were implanted prior to kindling. After 30 stimulations--delivered from days 4 to 8 after implantation--control animals had developed convulsive stage 5 seizures, whereas recipients of adenosine-releasing implants were still protected from convulsive seizures. Kindling was resumed after nine days to allow expiration of adenosine release. During additional 30 stimulations, recipients of adenosine-releasing implants gradually resumed kindling development at seizure stages corresponding to those when kindling was initially suspended, while control rats resumed kindling development at convulsive seizure stages. Blockade of adenosine A1 receptors did not exacerbate seizures in protected animals. We conclude that silk-based adenosine delivery exerts potent anti-ictogenic effects, but might also have at least partial anti-epileptogenic effects. Thus, silk-based adenosine augmentation holds promise for the treatment of epilepsy.

A device to study the initiation and propagation of calcium transients in cultured neurons after mechanical stretch

The brain is generally considered protected from mechanical forces. However, during traumatic events, cells in brain tissue are exposed to complex mechanical events including transient acceleration, pressure, and direct stretch. This paper presents a model to expose cultured cells of the central nervous system (CNS) to a defined stretch insult. The system is designed to apply a single transient uniaxial stretch to cultured cells; control of both the magnitude and rate of stretch allows study of a broad range of conditions, from physiologic to traumatic. Distinct from previous cell-stretching systems, we control the fraction of cells deformed and examine the response of stretched cells or the surrounding population of unstretched cells, the “mechanical penumbra,” of the same culture. Finally, we use this new model with cultured neurons, measuring the acute calcium response in traumatically stretched cells and the propagation of this calcium influx in neighboring, but unstretched cells. Stretched neurons exhibit a strain rate and magnitude-dependent response, unstretched exhibit an “all-or-none” response. This model will further our understanding of the complex interaction between mechanical stimuli, resulting biochemical cascades, and interaction between mechanical and other challenges, thereby furthering our understanding of the initiation and evolution of cellular damage following traumatic CNS injury.

Adenosine kinase determines the degree of brain injury after ischemic stroke in mice

Adenosine kinase (ADK) is the major negative metabolic regulator of the endogenous neuroprotectant and homeostatic bioenergetic network regulator adenosine. We used three independent experimental approaches to determine the role of ADK as a molecular target for predicting the brains susceptibility to ischemic stroke. First, when subjected to a middle cerebral artery occlusion model of focal cerebral ischemia, transgenic fb-Adk-def mice, which have increased ADK expression in striatum (164%) and reduced ADK expression in cortical forebrain (65%), demonstrate increased striatal infarct volume (126%) but almost complete protection of cortex (27%) compared with wild-type (WT) controls, indicating that cerebral injury levels directly correlate to levels of ADK in the CNS. Second, we demonstrate abrogation of lipopolysaccharide (LPS)-induced ischemic preconditioning in transgenic mice with brain-wide ADK overexpression (Adk-tg), indicating that ADK activity negatively regulates LPS-induced tolerance to stroke. Third, using adeno-associated virus-based vectors that carry Adk-sense or -antisense constructs to overexpress or knockdown ADK in vivo, we demonstrate increased (126%) or decreased (51%) infarct volume, respectively, 4 weeks after injection into the striatum of WT mice. Together, our data define ADK as a possible therapeutic target for modulating the degree of stroke-induced brain injury.

Ketogenic diet prevents epileptogenesis and disease progression in adult mice and rats.

Epilepsy is a highly prevalent seizure disorder which tends to progress in severity and become refractory to treatment. Yet no therapy is proven to halt disease progression or to prevent the development of epilepsy. Because a high fat low carbohydrate ketogenic diet (KD) augments adenosine signaling in the brain and because adenosine not only suppresses seizures but also affects epileptogenesis, we hypothesized that a ketogenic diet might prevent epileptogenesis through similar mechanisms. Here, we tested this hypothesis in two independent rodent models of epileptogenesis. Using a pentylenetetrazole kindling paradigm in mice, we first show that a KD, but not a conventional antiepileptic drug (valproic acid), suppressed kindling-epileptogenesis. Importantly, after treatment reversal, increased seizure thresholds were maintained in those animals kindled in the presence of a KD, but not in those kindled in the presence of valproic acid. Next, we tested whether a KD can halt disease progression in a clinically relevant model of progressive epilepsy. Epileptic rats that developed spontaneous recurrent seizures after a pilocarpine-induced status epilepticus were treated with a KD or control diet (CD). Whereas seizures progressed in severity and frequency in the CD-fed animals, KD-fed animals showed a prolonged reduction of seizures, which persisted after diet reversal. KD-treatment was associated with increased adenosine and decreased DNA methylation, the latter being maintained after diet discontinuation. Our findings demonstrate that a KD prevented disease progression in two mechanistically different models of epilepsy, and suggest an epigenetic mechanism underlying the therapeutic effects.

MicroRNA responses to focal cerebral ischemia in male and female mouse brain

Stroke occurs with greater frequency in men than in women across diverse ethnic backgrounds and nationalities. Work from our lab and others have revealed a sex-specific sensitivity to cerebral ischemia whereby males exhibit a larger extent of brain damage resulting from an ischemic event compared to females. Previous studies revealed that microRNA (miRNA) expression is regulated by cerebral ischemia in males; however, no studies to date have examined the effect of ischemia on miRNA responses in females. Thus, we examined miRNA responses in male and female brain in response to cerebral ischemia using miRNA arrays. These studies revealed that in male and female brains, ischemia leads to both a universal miRNA response as well as a sexually distinct response to challenge. Target prediction analysis of the miRNAs increased in male or female ischemic brain reveal sex-specific differences in gene targets and protein pathways. These data support that the mechanisms underlying sexually dimorphic responses to cerebral ischemia includes distinct changes in miRNAs in male and female brain, in addition to a miRNA signature response to ischemia that is common to both.