Amyotrophic lateral sclerosis and antibodies to voltage‐gated calcium channels—new doubts

Although the clinical and pathological features of amyotrophic lateral sclerosis (ALS) were described by Charcot more than 130 years ago, the underlying pathogenetic mechanisms remain unclear. It seems likely that motor neurons are particularly vulnerable to a variety of insults, any of which can trigger the pathways that ultimately lead to cell death. At present, the leading hypotheses include excitotoxic damage due to glutamate excess; genetic abnormalities (for instance in the superoxide dismutase [SOD-I] gene as found in some patients with familial HLS); and overproduction of neurofilaments, resulting in intracellular accumulation (see [ 11). Over the the past 25 years, the possibility of an autoimmune basis has also been considered (for a review, see [2]), and recently physiological and immunological evidence for antibodies to voltage-gated calcium channels in sera from ALS patients has been presented (see [3]). However, the role of autoimmunity has been questioned [ 4 ] , and now two reports in this issue [5 , 61 raise doubts about the existence of antibodies to calcium channels; one of them even suggests a nonimmunological explanation for some of the published observations. The calcium channel antibody hypothesis began with reports by Appel and his colleagues [3] at Baylor that immunoglobulin preparations (because some of the IgG preparations were only partially purified, by ammonium sulfate precipitation and ion-exchange chromatography, we refer to them as Ig, rather than IgG) from ALS patients increased miniature end-plate potential (MEPP) frequency at the neuromuscular junction (NMJ) of mice, both in vitro and after in vivo injection. Increases in MEPP frequency are usually associated with opening of the calcium channels at the motor nerve terminal, resulting in increased calcium influx. However, the Baylor group first studied calcium channels in skeletal muscle membrane preparations and showed, somewhat paradoxically, that Ig from ALS patients decreased calcium currents [3] . They then reported that ALS patients had increased levels of antibodies to L-type calcium channels, purified from rabbit muscle, as shown by enzyme-linked immunosorbent assay (ELISA), and that the levels correlated with the rapidity of disease progression [7]. Moreover, they subsequently reported binding of ALS Ig to purified a, subunits of these L-type channels on Western blots [S]. Calcium channels are a family of related proteins that have different localization and susceptibility to various blocking agents [9] . Skeletal muscle calcium channels are of the L type, but channels at motor nerve terminals are thought to be of the N, P, or P / Q type. Hence, these observations did not address the question of what ALS Ig preparations were doing at the neuromuscular junction. More consistent with the physiological evidence of increased MEPP frequency was the fact that two ALS Ig preparations increased P-type calcium currents in cerebellar Purkinje cells and increased the channel open time of purified P-type channels [ lo]. Moreover, Ig preparations from ALS patients induced death of a rat motor neuron/mouse neuroblastoma cell line, and the effects were prevented by blockade of Nor P-type channels [ I l l . Thus, at that point the findings were consistent with a stimulatory effect of ALS Ig on Nor P-type channels that might increase calcium uptake and be capable of causing death in susceptible neurons. Antibodies to calcium channels can also be measured by immunoprecipitation of '" I-neurotoxinlabeled channels, as has been successfully demonstrated in Lambert-Eaton myasthenic syndrome (LEMS) [ 12, 131. Although the Baylor group has not measured these antibodies in ALS sera, Lennon and coworkers [13] found antibodies to P/Q-type calcium channels in about 23% of ALS patients and antibodies to N-type channels in 13%, in both cases using specific ! ' 51 -~ conotoxins to label the channels. At that point one might have accepted the presence of the antibodies, at least in some patients, even if one is not convinced about their pathogenic relevance. However, we [4] have not been able to confirm the presence of antibodies to Nor P/Q-type channels in 26 ALS sera, and the two papers in this month's Annals of Neuroloa raise further questions about their existence. Arsac and colleagues [5] did not detect antibodies to either L-type or N-type calcium channels in ALS sera. They looked at precipitation of [ 'Hl-dihydropyridine-labeled I,-type channels from muscle and brain and found no positive results. It might be argued that the solid-state ELISA used by the Baylor group [7] was more efficient at binding low-affinity antibodies to Ltype channels than immunoprecipitation, which takes place in solution. T o test this contention, Arsac and colleagues [5] compared detection of antibodies in LEMS sera using immunoprecipitation of '"I-labeled N-type calcium channels with results using an ELISA. They found that the immunoprecipitation assay was slightly more sensitive (although this would not necessarily apply to immunoprecipitation of ['Hl-dihydropyridine-labeled L-type calcium channels). Neverthe-