Xiao-Ping He

Glutamate receptor GluR3 antibodies and death of cortical cells.

Rasmussens encephalitis (RE), a childhood disease characterized by epileptic seizures associated with progressive destruction of a single cerebral hemisphere, is an autoimmune disease in which one of the autoantigens is a glutamate receptor, GluR3. The improvement of some affected children following plasma exchange that removed circulating GluR3 antibodies (anti-GluR3) suggested that anti-GluR3 gained access to the central nervous system where it exerted deleterious effects. Here, we demonstrate that a subset of rabbits immunized with a GluR3 fusion protein develops a neurological disorder mimicking RE. Anti-GluR3 IgG isolated from serum of both ill and healthy GluR3-immunized animals promoted death of cultured cortical cells by a complement-dependent mechanism. IgG immunoreactivity decorated neurons and their processes in neocortex and hippocampus in ill but not in healthy rabbits. Moreover, both IgG and complement membrane attack complex (MAC) immunoreactivity was evident on neurons and their processes in the cortex of a subset of patients with RE. We suggest that access of IgG to epitopes in the central nervous system triggers complement-mediated neuronal damage and contributes to the pathogenesis of both this animal model and RE.

Transient Inhibition of TrkB Kinase after Status Epilepticus Prevents Development of Temporal Lobe Epilepsy

Temporal lobe epilepsy is the most common and often devastating form of human epilepsy. The molecular mechanism underlying the development of temporal lobe epilepsy remains largely unknown. Emerging evidence suggests that activation of the BDNF receptor TrkB promotes epileptogenesis caused by status epilepticus. We investigated a mouse model in which a brief episode of status epilepticus results in chronic recurrent seizures, anxiety-like behavior, and destruction of hippocampal neurons. We used a chemical-genetic approach to selectively inhibit activation of TrkB. We demonstrate that inhibition of TrkB commencing after status epilepticus and continued for 2 weeks prevents recurrent seizures, ameliorates anxiety-like behavior, and limits loss of hippocampal neurons when tested weeks to months later. That transient inhibition commencing after status epilepticus can prevent these long-lasting devastating consequences establishes TrkB signaling as an attractive target for developing preventive treatments of epilepsy in humans.

Autoimmunity to munc-18 in Rasmussen's encephalitis.

Rasmussens encephalitis (RE) is a rare disease of the central nervous system characterized by severe epileptic seizures, progressive degeneration of a single cerebral hemisphere, and autoimmunity directed against glutamate receptor subunit, GluR3. We report here the identification of high-titer autoantibodies directed against munc-18 in the serum of a single patient with RE previously shown to have anti-GluR3 antibodies. Munc-18 is an intracellular protein residing in presynaptic terminals, which is required for secretion of neurotransmitters. These findings are consistent with the possibility of intermolecular epitope spreading between GluR3, a postsynaptic cell surface protein, and munc-18, a presynaptic intracellular protein. Immune attack on these two proteins, which participate at distinct steps of synaptic transmission, could act in an additive or synergistic manner to impair synaptic function and lead to seizures and neuronal death.

Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy

We analyzed four families that presented with a similar condition characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. We show that recessive mutations in the ASNS gene are responsible for this syndrome. Two of the identified missense mutations dramatically reduce ASNS protein abundance, suggesting that the mutations cause loss of function. Hypomorphic Asns mutant mice have structural brain abnormalities, including enlarged ventricles and reduced cortical thickness, and show deficits in learning and memory mimicking aspects of the patient phenotype. ASNS encodes asparagine synthetase, which catalyzes the synthesis of asparagine from glutamine and aspartate. The neurological impairment resulting from ASNS deficiency may be explained by asparagine depletion in the brain or by accumulation of aspartate/glutamate leading to enhanced excitability and neuronal damage. Our study thus indicates that asparagine synthesis is essential for the development and function of the brain but not for that of other organs.

Structural Plasticity of Dentate Granule Cell Mossy Fibers During the Development of Limbic Epilepsy

Altered granule cell≫CA3 pyramidal cell synaptic connectivity may contribute to the development of limbic epilepsy. To explore this possibility, granule cell giant mossy fiber bouton plasticity was examined in the kindling and pilocarpine models of epilepsy using green fluorescent protein‐expressing transgenic mice. These studies revealed significant increases in the frequency of giant boutons with satellite boutons 2 days and 1 month after pilocarpine status epilepticus, and increases in giant bouton area at 1 month. Similar increases in giant bouton area were observed shortly after kindling. Finally, both models exhibited plasticity of mossy fiber giant bouton filopodia, which contact GABAergic interneurons mediating feedforward inhibition of CA3 pyramids. In the kindling model, however, all changes were fleeting, having resolved by 1 month after the last evoked seizure. Together, these findings demonstrate striking structural plasticity of granule cell mossy fiber synaptic terminal structure in two distinct models of adult limbic epileptogenesis. We suggest that these plasticities modify local connectivities between individual mossy fiber terminals and their targets, inhibitory interneurons, and CA3 pyramidal cells potentially altering the balance of excitation and inhibition during the development of epilepsy.

A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.

The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.

Morphological changes among hippocampal dentate granule cells exposed to early kindling-epileptogenesis.

Temporal lobe epilepsy is associated with changes in the morphology of hippocampal dentate granule cells. These changes are evident in numerous models that are associated with substantial neuron loss and spontaneous recurrent seizures. By contrast, previous studies have shown that in the kindling model, it is possible to administer a limited number of stimulations sufficient to produce a lifelong enhanced sensitivity to stimulus evoked seizures without associated spontaneous seizures and minimal neuronal loss. Here we examined whether stimulation of the amygdala sufficient to evoke five convulsive seizures (class IV or greater on Racines scale) produce morphological changes similar to those observed in models of epilepsy associated with substantial cell loss. The morphology of GFP‐expressing granule cells from Thy‐1 GFP mice was examined either 1 day or 1 month after the last evoked seizure. Interestingly, significant reductions in dendritic spine density were evident 1 day after the last seizure, the magnitude of which had diminished by 1 month. Further, there was an increase in the thickness of the granule cell layer 1 day after the last evoked seizure, which was absent a month later. We also observed an increase in the area of the proximal axon, which again returned to control levels a month later. No differences in the number of basal dendrites were detected at either time point. These findings demonstrate that the early stages of kindling epileptogenesis produce transient changes in the granule cell body layer thickness, molecular layer spine density, and axon proximal area, but do not produce striking rearrangements of granule cell structure.

Vagal nerve stimulation modifies neuronal activity and the proteome of excitatory synapses of amygdala/piriform cortex.

Vagal Nerve Stimulation (VNS) Therapy® is a United States Food and Drug Administration approved neurotherapeutic for medically refractory partial epilepsy and treatment‐resistant depression. The molecular mechanisms underlying its beneficial effects are unclear. We hypothesized that one mechanism involves neuronal activity‐dependent modifications of central nervous system excitatory synapses. To begin to test this hypothesis, we asked whether VNS modifies the activity of neurons in amygdala and hippocampus. Neuronal recordings from adult, freely moving rats revealed that activity in both amygdala and hippocampus was modified by VNS immediately after its application, and changes were detected following 1 week of stimulation. To investigate whether VNS modifies the proteome of excitatory synapses, we established a label‐free, quantitative liquid chromatography‐tandem mass spectrometry workflow that enables global analysis of the constituents of the postsynaptic density (PSD) proteome. PSD proteins were biochemically purified from amygdala/piriform cortex of VNS‐ or dummy‐treated rats following 1‐week stimulation, and individual PSD protein levels were quantified by liquid chromatography‐tandem mass spectrometry analysis. We identified 1899 unique peptides corresponding to 425 proteins in PSD fractions, of which expression levels of 22 proteins were differentially regulated by VNS with changes greater than 150%. Changes in a subset of these proteins, including significantly increased expression of neurexin‐1α, cadherin 13 and voltage‐dependent calcium channel α2δ1, the primary target of the antiepileptic drug gabapentin, and decreased expression of voltage‐dependent calcium channel γ3, were confirmed by western blot analysis of PSD samples. These results demonstrate that VNS modulates excitatory synapses through regulating a subset of the PSD proteome. Our study reveals molecular targets of VNS and point to possible mechanisms underlying its beneficial effects, including activity‐dependent formation of excitatory synapses.

Conditional Deletion of TrkB Prevents Epileptogenesis in the Kindling Model

Epilepsy is a common and frequently devastating neurological disorder, affecting approximately 1% of the population. Among the diverse forms, limbic epilepsy (synonyms include complex partial epilepsy, temporal lobe epilepsy, psychomotor epilepsy) in particular is the most devastating in adults for three reasons: (1) it is common, accounting for approximately 40% of all cases of adult epilepsy; (2) limbic seizures are often quite resistant to available anticonvulsant drugs;1 and (3) the attacks induce impairment of consciousness, thereby limiting driving, maintaining employment, etc. Therapy is only symptomatic in that available drugs inhibit seizures in some individuals but do not modify the disease itself.