TD-12 workshop report: characterization of monoclonal antibodies to neuron-specific enolase.

Twelve antibodies to neuron-specific enolase (NSE) have been evaluated by four working groups. Human brain γγ-enolase, neuroblastoma-derived αγ-enolase, and recombinant γγ-enolase were used to determine antibody specificity and binding kinetics. All antibodies were found to be specific for the γ-isoform. It was possible to assign 11 of the antibodies to at least five epitope groups based on cross-inhibition experiments, QCM and SPR technology, and immunoassay combinations. Antibodies 9601 and 9602 showed the highest affinity for both native and recombinant γγ-enolase. Immunometric assays for both γγ- and αγ-enolase could be made by pairing 9601 with most of the other ISOBM antibodies. Antibodies differed in their ability to recognize native αγ-enolase, native γγ-enolase, and recombinant γγ-enolase. Some immunometric assay combinations appear to favor the detection of heterodimeric αγ-enolase over the homodimeric γγ-enolase. Although the majority of the antibodies failed to detect human NSE or recombinant NSE in Western blots, mAb 9601 recognized both, while E17 and 18E5 were specific for human NSE.

Hyperekplexia phenotype of glycine receptor alpha1 subunit mutant mice identifies Zn(2+) as an essential endogenous modulator of glycinergic neurotransmission.

Zn(2+) is thought to modulate neurotransmission by affecting currents mediated by ligand-gated ion channels and transmitter reuptake by Na(+)-dependent transporter systems. Here, we examined the in vivo relevance of Zn(2+) neuromodulation by producing knockin mice carrying the mutation D80A in the glycine receptor (GlyR) alpha1 subunit gene (Glra1). This substitution selectively eliminates the potentiating effect of Zn(2+) on GlyR currents. Mice homozygous for Glra1(D80A) develop a severe neuromotor phenotype postnatally that resembles forms of human hyperekplexia (startle disease) caused by mutations in GlyR genes. In spinal neurons and brainstem slices from Glra1(D80A) mice, GlyR expression, synaptic localization, and basal glycinergic transmission were normal; however, potentiation of spontaneous glycinergic currents by Zn(2+) was significantly impaired. Thus, the hyperekplexia phenotype of Glra1(D80A) mice is due to the loss of Zn(2+) potentiation of alpha1 subunit containing GlyRs, indicating that synaptic Zn(2+) is essential for proper in vivo functioning of glycinergic neurotransmission.

A re-engineered immunotoxin shows promising preclinical activity in ovarian cancer.

RG7787 is a re-engineered mesothelin-targeted immunotoxin with reduced immunogenicity composed of a humanized anti-mesothelin Fab fragment and a B-cell epitope silenced 24 kD fragment of Pseudomonas exotoxin A. High prevalence of mesothelin-positive cases and a large unmet medical need make ovarian cancer a promising indication for the clinical development of RG7787. However, ovarian cancer patients also frequently have elevated serum levels of the cancer antigen 125 (CA-125). In principle this could pose a problem, since the binding sites for CA-125 and RG7787 on mesothelin were reported to overlap. However, we show here that RG7787 can readily displace even excess amounts of CA-125 in different cellular assays. Moreover when tested in-vitro on a panel of 12 ovarian cancer cell lines, RG7787 had high cytotoxic activity on COV644, Caov-4, and SNU-119 cells and fully inhibited growth of EFO-21, KURAMOCHI, OVSAHO, and Caov-3 cells with potency values ranging from 1 to 86 pM. Finally, we evaluated the in-vivo efficacy of RG7787 in OvCa6668, a patient-derived ovarian cancer model with high levels of CA-125 expression. RG7787 had moderate monotherapy efficacy but in combination with standard chemotherapies (cisplatin, paclitaxel) achieved pronounced tumor regressions. In summary our data support clinical testing of RG7787 in ovarian cancer.

Matching molecules to function: neuronal Ca2+-activated K+ channels and afterhyperpolarizations.

Potassium channels regulate the membrane excitability of neurons, play a major role in shaping action potentials, determining firing patterns and regulating neurotransmitter release, and thus significantly contribute to neuronal signal encoding and integration. This review focuses on the molecular and cellular basis for the specific function of small-conductance calcium-activated potassium channels (SK channels) in the nervous system. SK channels are activated by an intracellular increase of free calcium during action potentials. They mediate currents that modulate the firing frequency of neurons. Three SK channel subunits have been cloned and form channels, which are voltage-insensitive, activated by submicromolar intracellular calcium concentrations, and are blocked, with different affinities, by a number of toxins and organic compounds. Different neurons in the central and peripheral nervous system express distinct subsets of SK channel subunits. Recent progress has been made in relating cloned SK channels to their native counterparts. These findings argue in favour of regulatory mechanisms conferring to native SK channels with specific subunit compositions distinct and specific functional profiles in different neurons.

Domain analysis of the calcium-activated potassium channel SK1 from rat brain. Functional expression and toxin sensitivity.

Two small conductance, calcium-activated potassium channels (SK channels), SK2 and SK3, have been shown to contribute to the afterhyperpolarization (AHP) and to shape the firing behavior in neurons for example in the hippocampal formation, the dorsal vagal nucleus, the subthalamic nucleus, and the cerebellum. In heterologous expression systems, SK2 and SK3 currents are blocked by the bee venom toxin apamin, just as well as the corresponding neuronal AHP currents. However, the functional role and pharmacological profile of SK1 channels from rat brain (rSK1) is still largely unknown, as so far rSK1 homomeric channels could not be functionally expressed. We have performed a domain analysis to elucidate the pharmacological profile and the molecular determinants of rSK1 channel expression by using channel chimeras in combination with immunocytochemistry, immunoblot analysis, and electrophysiology. Our results reveal that the rSK1 subunit is synthesized in cells but does not form functional homomeric channels. Exchanging the carboxyl terminus of rSK1 for that of hSK1 or rSK2 is sufficient to rescue the functional expression of rSK1 channels. Additionally, transplantation of both amino and carboxyl termini of rSK1 onto hSK1 subunits, normally forming functional homomeric channel, hinders their functional expression, while hSK1 channels containing only the rSK1 carboxyl terminus are functional. These results suggest that the lack of functional expression of rSK1 channels is probably due to problems in their assembly and tetramerization but not in their calmodulin-dependent gating. Finally, we show that chimeric channels containing the core domain (S1-S6) of rSK1, unlike hSK1, are apamin-insensitive.