Trophic factor BDNF inhibits GABAergic signaling by facilitating dendritic enrichment of SUMO E3 ligase PIAS3 and altering gephyrin scaffold.

Posttranslational addition of a small ubiquitin-like modifier (SUMO) moiety (SUMOylation) has been implicated in pathologies such as brain ischemia, diabetic peripheral neuropathy, and neurodegeneration. However, nuclear enrichment of SUMO pathway proteins has made it difficult to ascertain how ion channels, proteins that are typically localized to and function at the plasma membrane, and mitochondria are SUMOylated. Here, we report that the trophic factor, brain-derived neurotrophic factor (BDNF) regulates SUMO proteins both spatially and temporally in neurons. We show that BDNF signaling via the receptor tropomyosin-related kinase B facilitates nuclear exodus of SUMO proteins and subsequent enrichment within dendrites. Of the various SUMO E3 ligases, we found that PIAS-3 dendrite enrichment in response to BDNF signaling specifically modulates subsequent ERK1/2 kinase pathway signaling. In addition, we found the PIAS-3 RING and Ser/Thr domains, albeit in opposing manners, functionally inhibit GABA-mediated inhibition. Finally, using oxygen-glucose deprivation as an in vitro model for ischemia, we show that BDNF-tropomyosin-related kinase B signaling negatively impairs clustering of the main scaffolding protein at GABAergic postsynapse, gephyrin, whereby reducing GABAergic neurotransmission postischemia. SUMOylation-defective gephyrin K148R/K724R mutant transgene expression reversed these ischemia-induced changes in gephyrin cluster density. Taken together, these data suggest that BDNF signaling facilitates the temporal relocation of nuclear-enriched SUMO proteins to dendrites to influence postsynaptic protein SUMOylation.

Prolonged ampakine exposure prunes dendritic spines and increases presynaptic release probability for enhanced long-term potentiation in the hippocampus.

CX 546, an allosteric positive modulator of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic glutamate receptors (AMPARs), belongs to a drug class called ampakines. These compounds have been shown to enhance long-term potentiation (LTP), a cellular model of learning and memory, and improve animal learning task performance, and have augmented cognition in neurodegenerative patients. However, the chronic effect of CX546 on synaptic structures has not been examined. The structure and integrity of dendritic spines are thought to play a role in learning and memory, and their abnormalities have been implicated in cognitive disorders. In addition, their structural plasticity has been shown to be important for cognitive function, such that dendritic spine remodeling has been proposed as the morphological correlate for LTP. Here, we tested the effect of CX546 on dendritic spine remodeling following long-term treatment. We found that, with prolonged CX546 treatment, organotypic hippocampal slice cultures showed a significant reduction in CA3-CA1 excitatory synapse and spine density. Electrophysiological approaches revealed that the CA3-CA1 circuitry compensates for this synapse loss by increasing synaptic efficacy through enhancement of presynaptic release probability. CX546-treated slices showed prolonged and enhanced potentiation upon LTP induction. Furthermore, structural plasticity, namely spine head enlargement, was also more pronounced after CX546 treatment. Our results suggest a concordance of functional and structural changes that is enhanced with prolonged CX546 exposure. Thus, the improved cognitive ability of patients receiving ampakine treatment may result from the priming of synapses through increases in the structural plasticity and functional reliability of hippocampal synapses.

Blocking brain-derived neurotrophic factor inhibits injury-induced hyperexcitability of hippocampal CA3 neurons.

Brain trauma can disrupt synaptic connections, and this in turn can prompt axons to sprout and form new connections. If these new axonal connections are aberrant, hyperexcitability can result. It has been shown that ablating tropomyosin-related kinase B (TrkB), a receptor for brain-derived neurotrophic factor (BDNF), can reduce axonal sprouting after hippocampal injury. However, it is unknown whether inhibiting BDNF-mediated axonal sprouting will reduce hyperexcitability. Given this, our purpose here was to determine whether pharmacologically blocking BDNF inhibits hyperexcitability after injury-induced axonal sprouting in the hippocampus. To induce injury, we made Schaffer collateral lesions in organotypic hippocampal slice cultures. As reported by others, we observed a 50% reduction in axonal sprouting in cultures treated with a BDNF blocker (TrkB-Fc) 14 days after injury. Furthermore, lesioned cultures treated with TrkB-Fc were less hyperexcitable than lesioned untreated cultures. Using electrophysiology, we observed a two-fold decrease in the number of CA3 neurons that showed bursting responses after lesion with TrkB-Fc treatment, whereas we found no change in intrinsic neuronal firing properties. Finally, evoked field excitatory postsynaptic potential recordings indicated an increase in network activity within area CA3 after lesion, which was prevented with chronic TrkB-Fc treatment. Taken together, our results demonstrate that blocking BDNF attenuates injury-induced hyperexcitability of hippocampal CA3 neurons. Axonal sprouting has been found in patients with post-traumatic epilepsy. Therefore, our data suggest that blocking the BDNF-TrkB signaling cascade shortly after injury may be a potential therapeutic target for the treatment of post-traumatic epilepsy.

Cross-talk between GABAergic postsynapse and microglia regulate synapse loss after brain ischemia.

Microglia interact with neurons to facilitate synapse plasticity; however, signal(s) contributing to microglia activation for synapse elimination in pathology are not fully understood. Here, using in vitro organotypic hippocampal slice cultures and transient middle cerebral artery occlusion (MCAO) in genetically engineered mice in vivo, we report that at 24 hours after ischemia, microglia release brain-derived neurotrophic factor (BDNF) to downregulate glutamatergic and GABAergic synapses within the peri-infarct area. Analysis of the cornu ammonis 1 (CA1) in vitro shows that proBDNF and mBDNF downregulate glutamatergic dendritic spines and gephyrin scaffold stability through p75 neurotrophin receptor (p75NTR) and tropomyosin receptor kinase B (TrkB) receptors, respectively. After MCAO, we report that in the peri-infarct area and in the corresponding contralateral hemisphere, similar neuroplasticity occurs through microglia activation and gephyrin phosphorylation at serine-268 and serine-270 in vivo. Targeted deletion of the Bdnf gene in microglia or GphnS268A/S270A (phospho-null) point mutations protects against ischemic brain damage, neuroinflammation, and synapse downregulation after MCAO.

Chamber and microfluidic probe for microperfusion of organotypic brain slices.

Microfluidic systems are increasingly being used for the culture and study of dissociated cells because they require only minute amounts of materials while enabling drug screening and chemotaxis studies down to the single cell level. However, the culture of organized tissue, such as brain slices, has been more difficult to adapt to microfluidic devices. Here, we present a microfluidic system, comprising (i) a perfusion chamber for the culture of organotypic slices that is compatible with high resolution imaging on inverted microscopes, and (ii) a novel transparent microfluidic probe (MFP) for the localized microperfusion of the brain tissue. The MFP is made in poly(dimethylsiloxane), features six micrometre-scale apertures and can be assembled within a few hours in a standard laboratory. Each aperture can indiscriminately be used either for the injection or aspiration of solutions, giving rise to many possible combinations. The MFP was successfully used for the perfusion of a small number of cells in a brain slice with concurrent confocal fluorescence imaging of the perfused dye and sub-cellular structures within the tissue.

DNA methylation mediates persistent epileptiform activity in vitro and in vivo.

Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5' regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5' region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5' region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5' region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5' region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.