Ephrin-B2 prevents N-methyl-D-aspartate receptor antibody effects on memory and neuroplasticity.

OBJECTIVE: To demonstrate that ephrin-B2 (the ligand of EphB2 receptor) antagonizes the pathogenic effects of patients' N-methyl-D-aspartate receptor (NMDAR) antibodies on memory and synaptic plasticity. METHODS: One hundred twenty-two C57BL/6J mice infused with cerebrospinal fluid (CSF) from patients with anti-NMDAR encephalitis or controls, with or without ephrin-B2, were investigated. CSF was infused through ventricular catheters connected to subcutaneous osmotic pumps over 14 days. Memory, behavioral tasks, locomotor activity, presence of human antibodies specifically bound to hippocampal NMDAR, and antibody effects on the density of cell-surface and synaptic NMDAR and EphB2 were examined at different time points using reported techniques. Short- and long-term synaptic plasticity were determined in acute brain sections; the Schaffer collateral pathway was stimulated and the field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. RESULTS: Mice infused with patients' CSF, but not control CSF, developed progressive memory deficit and depressive-like behavior along with deposits of NMDAR antibodies in the hippocampus. These findings were associated with a decrease of the density of cell-surface and synaptic NMDAR and EphB2, and marked impairment of long-term synaptic plasticity without altering short-term plasticity. Administration of ephrin-B2 prevented the pathogenic effects of the antibodies in all the investigated paradigms assessing memory, depressive-like behavior, density of cell-surface and synaptic NMDAR and EphB2, and long-term synaptic plasticity. INTERPRETATION: Administration of ephrin-B2 prevents the pathogenic effects of anti-NMDAR encephalitis antibodies on memory and behavior, levels of cell-surface NMDAR, and synaptic plasticity. These findings reveal a strategy beyond immunotherapy to antagonize patients' antibody effects. Ann Neurol 2016;80:388-400.

Longitudinal optic neuritis-unrelated visual evoked potential changes in NMO spectrum disorders.

OBJECTIVE: To investigate if patients with neuromyelitis optica spectrum disorder (NMOSD) develop subclinical visual pathway impairment independent of acute attacks. METHODS: A total of 548 longitudinally assessed full-field visual evoked potentials (VEP) of 167 patients with NMOSD from 16 centers were retrospectively evaluated for changes of P100 latencies and P100-N140 amplitudes. Rates of change in latencies (RCL) and amplitudes (RCA) over time were analyzed for each individual eye using linear regression and compared using generalized estimating equation models. RESULTS: The rates of change in the absence of optic neuritis (ON) for minimal VEP intervals of ≥3 months between baseline and last follow-up were +1.951 ms/y (n = 101 eyes; SD = 6.274; p = 0.012) for the P100 latencies and -2.149 µV/y (n = 64 eyes; SD = 5.013; p = 0.005) for the P100-N140 amplitudes. For minimal VEP intervals of ≥12 months, the RCL was +1.768 ms/y (n = 59 eyes; SD = 4.558; p = 0.024) and the RCA was -0.527 µV/y (n = 44 eyes; SD = 2.123; p = 0.111). The history of a previous ON >6 months before baseline VEP had no influence on RCL and RCA. ONs during the observational period led to mean RCL and RCA of +11.689 ms/y (n = 16 eyes; SD = 17.593; p = 0.003) and -1.238 µV/y (n = 11 eyes; SD = 3.708; p = 0.308), respectively. CONCLUSION: This first longitudinal VEP study of patients with NMOSD provides evidence of progressive VEP latency delay occurring independently of acute ON. Prospective longitudinal studies are needed to corroborate these findings and help to interpret the clinical relevance.

Interactions of Human Autoantibodies with Hippocampal GABAergic Synaptic Transmission - Analyzing Antibody-Induced Effects ex vivo.

Autoantibodies (aAB) to the presynaptic located enzyme glutamate decarboxylase 65 (GAD65) are a characteristic attribute for a variety of autoimmune diseases of the central nervous system including subtypes of limbic encephalitis, stiff person-syndrome, cerebellar ataxia, and Batten's disease. Clinical signs of hyperexcitability and improvement of disease symptoms upon immunotherapy in some of these disorders suggest a possible pathogenic role of associated aAB. Recent experimental studies report inconsistent results regarding a direct pathogenic influence of anti-GAD65 aAB affecting inhibitory synaptic transmission in central GABAergic pathways. We here provide a method for direct evaluation of aAB-induced pathomechanisms in the intact hippocampal network. Purified patient IgG fractions containing aAB to GAD65 together with fixable lipophilic styryl dyes (FMdyes) are stereotactically injected into the hilus and the dentate gyrus in anesthetized mice. Twenty-four hours after intrahippocampal injection, acute hippocampal slices are prepared and transferred to a patch-clamp recording setup equipped with a fluorescence light source. Intraneural incorporated FMdyes show correct injection site for patch-clamp recording. Whole-cell patch-clamp recordings are performed from granule cells in the dentate gyrus and extracellular stimulation is applied in the border area of the dentate gyrus-hilus region to stimulate GABAergic afferents arising from parvalbumin positive basket cells. GABA-A receptor mediated inhibitory postsynaptic currents (IPSC) and miniature IPSC are recorded after blocking glutamatergic transmission. This approach allows investigation of potential aAB-induced effects on GABA-A receptor signaling ex vivo in an intact neuronal network. This offers several advantages compared to experimental procedures used in previous studies by in vitro AB preincubation of primary neurons or slice preparations. Furthermore, this method requires only small amounts of patient material that are often limited in rare diseases.

Targeting PI3K in neuroblastoma.

PURPOSE: This work employs pharmacological targeting of phosphoinositide 3-kinases (PI3K) in selected neuroblastoma (NB) tumors with the inhibitor AS605240, which has been shown to express low toxicity and relative specificity for the PI3K species γ. METHODS: The expression pattern of PI3K isoforms in 7 NB cell lines and 14 tumor patient samples was determined by Western blotting and immunocytochemistry. The effect of AS605240 on the growth of four selected tumor cell lines was assessed. Two cell lines exhibiting (SK-N-LO) or lacking (SK-N-AS) PI3Kγ expression were chosen for further in vitro analysis, which involved propidium iodide (PI)-based cell cycle staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL-staining) of apoptotic cells and analysis of PI3K/Akt-related signaling pathways via Western blotting and translocation experiments. The action of AS605240 in vivo was addressed by xenograft experiments in severe combined immunodeficiency (SCID) mice, thereby comparing SK-N-LO and SK-N-AS derived tumors. Apoptosis induced in SK-N-LO tumors was shown by immunohistochemical TUNEL-staining. RESULTS: Significant expression of PI3Kγ in neuroblastoma patient biopsies and tumor cell lines was detected. AS605240 induced apoptosis in NB cell lines proportional to this expression and suppressed growth of PI3Kγ positive, but not negative, tumors in a xenograft mouse model. No adverse effects of the inhibitor treatment were observed. CONCLUSIONS: Our observations hint to an oncogenic function of PI3Kγ in distinct neuroblastoma entities and reveal PI3K targeting by AS605240 as a promising molecular therapy of these tumors.