Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive neuromuscular dysfunction and decrease in the number of upper and lower motoneurons (Mulder 1982). Clinical manifestations include fatigue, fasciculations, spasticity, hyperreflexia, weakness and muscle atrophy, ultimately leading to paralysis and death (Rowland 1998). Currently, there are no effective treatments to either stop or delay ALS progression.
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In the present study, we demonstrated the existence of a subpopulation of sALS patients (63% in our sample), whose IgG fractions induced a significant enhancement of spontaneous acetylcholine release (Fig. 1). A similar effect has been postulated to be the consequence of increased intracellular Ca2+ at nerve terminals caused by ALS–IgG binding to targets in presynaptic membranes (Uchitel et al. 1988, 1992b; Pagani et al. 2006). IgGs from the same set of sALS patients also exhibited strong immunolabeling in wild-type mouse NMJs (Figs 2 and 3). Together, our results reinforce those of Pagani et al. (2006) who found an association between ALS–IgG binding and electrophysiological effects. From our data, it is also apparent that it is possible to separate sALS patients into two groups; one of which appears to have a strong autoimmune, neuromuscular-related component and that might contribute towards disease neuropathology.
There is a growing body of evidence supporting a role for Ca2+-dependent signaling as an important regulator of cellular dysfunction and death (Appel et al. 2001; Orrenius et al. 2003). Furthermore, spinal motoneurons are known to be particularly vulnerable to Ca2+ imbalance caused by their diminished expression of Ca2+-buffering proteins (Alexianu et al. 1994). An increase in intracellular Ca2+ levels above a certain threshold is predicted to trigger processes such as calpain activation, protein misfolding-endoplasmic reticulum stress, reactive oxygen species production and glutamate release (Orrenius et al. 2003; Smaili et al. 2009). The stimulation of any of these pathways via an aberrant rise in intracellular Ca2+ concentration could lead to the induction of apoptosis and provide a key link underlying ALS pathology. Additionally, reports documenting an enhancement in Ca2+ content of motoneurons and synaptic terminals in mice injected with ALS–IgGs have provided a key connection between Ca2+ overload and ALS (Engelhardt et al. 1995; Pullen and Humphreys 2000).
The comparison of ALS–IgG reactivity levels against NMJ (Fig. 3) and their potentiator effect on end-plate basal discharge activity (Fig. 1) indicates that both characteristics show a basically similar behavior, even though their sensitivities seem to be moderately different. Apart from differences in the intrinsic sensitivity of each technique employed, the discrepancy might also arrive as a consequence of the presence in some samples of several antibodies capable of interacting with the motor terminals.
The presence of significant levels of immunoreactivity in some disease controls was not unexpected (Figs 2–5 and Figures S1 and S2), as these individuals might have antibodies against proteins involved in synaptic transmission (i.e. voltage-dependent Ca2+ channels in DC1 and acetylcholine receptors in DC2). In this way, these samples serve as useful positive controls for the immunohistochemical techniques used to detect autoantibodies in sALS samples. Another important aspect to consider is the complete lack of reactivity in all the fALS samples analyzed (Fig. 3), as well as their inability to affect end-plate spontaneous synaptic activity (Fig. 1b). These results emphasize the specificity of our findings concerning sALS immunoglobulins since it is known that the hereditary form of the disease is clinically indistinguishable from the sporadic variant.
The finding that the IgG sample from DC1 (a Lambert-Eaton myasthenic syndrome patient) exhibited a similar level of interaction with CaV2.1+/+ and CaV2.1−/− end-plates might seem contradictory at first sight, because of the proposed etiology for this disease. However, it should be stressed that this sample might contain antibodies against not only CaV2.1 Ca2+ channels but also CaV2.2 (Takamori et al. 1995; Motomura et al. 1997). This latter channel is known to compensate for the lack of P/Q-type channels, both at the functional (Urbano et al. 2003) and proteic level (Pagani et al. 2004), and therefore could explain the invariability observed in immunostaining.
To further characterize ALS antibody reactivity against other neuronal substrates possibly relevant to disease pathophysiology, we performed immunofluorescence assays on mouse spinal and brain cortical slices. These neural structures are the site of residence for lower and upper motoneurons, respectively, both of which are important movement effectors. Cerebellar structures were also analyzed since they comprise cells involved in movement coordination and it has been shown that ALS antibodies are capable of modulating P/Q-type Ca2+ currents (Llinas et al. 1993). We found a broad reactivity pattern for ALS–IgGs in neuronal tissues such as the cerebellum and spinal cord (Figure S1), in which the vast majority of samples analyzed resulted in positive staining. Of note, we could not detect any difference in the reactivity of ALS–IgGs when comparing WT to CaV2.1-null animals in these regions (Figure S2). These data are in striking contrast to the clear differences detected in immunofluorescence experiments performed on NMJs (Fig. 4), and thus highlight the specificity of the findings for end-plate preparations. Along these lines, we speculate that the antigen(s) responsible for ALS–IgG labeling motor terminals is(are) predominantly expressed in these more peripheral synaptic sites than others such as the cerebellum or spinal ventral horn. However, we cannot exclude the possibility that the presence of the ALS–IgG reactive antigen(s) could be masked by other antigens expressed in the cerebellar or spinal cord neurons and which are more abundant or reactive towards ALS–IgGs. Additionally, the presence in ALS sera of such CNS-reactive antibodies is more likely the consequence of intracellular content exposure caused by neuronal injury, owing to their broad distribution amongst several ALS samples and the lack of correlation with NMJ binding.
As P/Q-type channels are the principal mediators of synaptic transmission in mammalian NMJs (Uchitel et al. 1992a; Katz et al. 1997) and there is evidence supporting an interaction between ALS–IgGs and Ca2+ channels (Llinas et al. 1993; Smith et al. 1994; Carter and Mynlieff 2003), we analyzed the effects of ALS–IgGs on deleting the CaV2.1 subunit. Our results showed that the absence of the P/Q-type channel CaV2.1 subunit produced a significant decrease in ALS–IgG binding to mouse NMJs (Fig. 4), as well as the complete suppression of ALS–IgG-mediated effects on enhancing spontaneous acetylcholine release (Fig. 6). Taken together, these data suggested that a subset of ALS–IgGs interact with either the CaV2.1 subunit or other protein(s) whose expression in NMJs is dramatically diminished as a consequence of CaV2.1 subunit deletion. In support of the latter, it is known that CaV2.1 subunit-null mice exhibit alterations in the expression of other genes besides the CaV2.1 subunit (Piedras-Renteria et al. 2004). Furthermore, other reports suggest that Ca2+ influx through P/Q-type channels – and hence, their presence and functionality – might be an important factor relevant to the transcription of synapse-related genes such as the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein syntaxin 1A (Sutton et al. 1999; Barbado et al. 2009).
A microarray analysis of the CaV2.1 gene (CACNA1A) expression profile revealed a seven-fold decrease in CaV2.1 messenger ribonucleic acid levels in rat extraocular muscle compared with a typical skeletal muscle such as tibialis anterior (Fischer et al. 2002). The extraocular muscle is well-known for being relatively spared during ALS progression (Leveille et al. 1982) and has been reported to be resistant to ALS–IgG NMJ effects in passive transfer experiments (Mosier et al. 2000). These studies provide a further important link between P/Q-type channel expression and ALS susceptibility.
Distinct from the above arguments in favor of a direct role of functional P/Q-type channels, our immunoprecipitation and western blot experiments using HEK293 cells expressing cloned P/Q-type channel subunits do not support a direct interaction of ALS–IgGs with the channel complex itself (Figure S3), at least under the conditions analyzed for this study. In support, ALS–IgGs have been shown not to interact with 125I-ω-conotoxin MVIIC-labeled channels (Drachman et al. 1995) and also that P/Q-type channel inhibition by ω-agatoxin IVA did not prevent immunoglobulin-induced synaptic potentiation (Pagani et al. 2006). Taken together, our results suggest that the protein(s) interacting with ALS–IgGs requires CaV2.1 expression/activity at the NMJ although it is CaV2.1-expression independent in other neuronal structures such as the cerebellum and spinal cord.
Another aspect worth noting is the lack of change in antibody binding to CaV2.1−/− NMJ for disease control sera compared to WT conditions (Fig. 4b). This invariability in immunostaining adds relevance to ALS–IgG findings by highlighting their specificity towards a selected subset of patients.
N-type Ca2+ channel activation has been found to be relevant for the induction of spontaneous synaptic activity by ALS–IgGs (Pagani et al. 2006). However, this effect is unlikely the consequence of a direct interaction between antibodies and the CaV2.2 N-type subunit as the loss of CaV2.2 did not significantly alter IgG binding of most of the ALS samples at mouse NMJs (Fig. 5). Moreover, it has been shown that in CaV2.1-null mutant animals, there is a functional compensation in neuromuscular transmission by N-type Ca2+ channels (Urbano et al. 2003), as well as an increment in channel protein levels (Pagani et al. 2004). Therefore, this could explain the significant changes in ALS–IgG immunoreactivity noted here (Fig. 4). Together, these results do not support a direct binding of ALS antibodies to the N-type channel but rather an indirect effect of IgGs on channel activity, for example through a second messenger signaling pathway, as it has been extensively reported for this channel (Lipscombe et al. 1989; Werz et al. 1993; Martin et al. 2006). In fact, Pagani et al. (2006) did detect a requirement of phospholipase C, ryanodine receptor and inositol triphosphate receptor activation for the ALS–IgG-induced synaptic potentiation to occur.
Several reports have documented the presence of human antibodies at the motoneuronal soma and nerve terminals of mice injected with ALS–IgGs (Fratantoni et al. 1996; Engelhardt et al. 2005), we speculated it might be the consequence of an interaction between disease antibodies and pre-synaptic components. In order to test this possibility, we assessed by immunoprecipitation experiments ALS–IgGs binding to several pre-synaptic proteins purified from synaptosomal extracts. We found that none of the evaluated ALS samples showed affinity towards proteins present at synaptic terminals, such as synaptotagmin I, syntaxin 1A, synaptophysin and synaptobrevin II, all of which were readily detectable in the original lysates (Figure S3f). Other candidates to test in the future include extracellular matrix molecules, neurotrophins receptors and proteins involved in synaptogenesis and axon guidance, due to their proposed role during neurodegeneration (Dawbarn and Allen 2003; Glas et al. 2007; Schmidt et al. 2009).
The results in the current work add relevant evidence in favor of autoimmunity as one of the possible mechanisms contributing to ALS pathology. They also suggest for the first time that, as opposed to generally interacting with all central and peripheral P/Q-type Ca2+ channels, autoantibodies in the serum of a subset of patients selectively interact with antigens at NMJs. The results also contribute towards further studies aimed at defining the IgGs as biological markers for sALS and in disease pathophysiology.
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Figure S1. Immunofluorescence of Swiss mice (P > 30) cerebellum and spinal cord slides (150 μm) incubated with purified IgGs from HC, DC or ALS. Presence of primary antibody was revealed by using an anti-human IgG attached to FITC. A) Representative confocal micrographs of Purkinje neurons. Bar: 25 μm. M and G stand for molecular and granular cerebellar layers, respectively. B) Fluorescence intensity determinations normalized to isotype control. Statistical analysis was performed by Rank Sum test. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to HCs. Figures inside bars indicate the number of cells studied in each experimental condition (N = 7 animals). C) Representative images of ventral horn-residing motoneurons. Bar: 25 μm. D) Fluorescence intensity quantification normalized to isotype control. Statistical analysis was performed by Rank Sum test. * p < 0.01 and ** p < 0.001 compared to HCs. Figures inside bars indicate the number of cells studied in each experimental condition (N = 5 animals).
Figure S2. Immunofluorescence of mouse cerebellum and spinal cord slides (150 μm) from WT or CaV2.1−/− mice (P16-23), incubated with purified IgGs from HC, DC or ALS. Detection of primary antibody was performed as previously described. A) Representative confocal micrographs of Purkinje neurons. Bar: 25 μm. B) Fluorescence intensity quantification normalized to isotype control. Statistical analysis between WT and CaV2.1-null preparations was performed by Two-way anova. * p < 0.05 and ** p < 0.001 compared to WT. The number of cells analyzed in each experimental condition varied between 15 and 39 (N = 5 animals). C) Representative confocal micrographs of ventral horn-residing motoneurons. Bar: 25 μm. D) Fluorescence intensity quantification normalized to isotype control. Statistical analysis between WT and CaV2.1-null preparations was performed by Two-way anova. * p < 0.01 and ** p < 0.001 compared to WT. The number of cells analyzed in each experimental condition varied between 15 and 24 (N = 4 animals).
Figure S3. Interaction of ALS antibodies with proteins from P/Q-type channel- expressing cells. Cultures express either the auxiliary subunits alone (β2a and α2δ2, -) or the intact P/Q-type channel (CaV2.1, β2a and α2δ2, +). Interactions with ALS antibodies were assayed 36 h post-transfection/induction. A) and B) Western blot of lysates from transfected (+,−) or non-transfected (NT) HEK293 cells. Blots were incubated either with ALS antibodies (1:500) or with a rabbit, polyclonal, anti-CaV2.1 IgG (1:10000) as a positive control. In B, a different part from the blots shown in A is depicted. C) Immunoprecipitation assay of transfected HEK293 cells using either ALS-IgGs or an anti- CaV2.1 antibody as a positive control. An aliquot of the original lysate was run in the same gel to check for the correct expression of the CaV2.1 subunit (input). A negative control by omission of antibodies was also included (beads). D) Western blot of lysates from T-rex 293 cells exposed (+) or not (-) to the inductor agent Tetracycline for 48 h. Membranes were incubated either with ALS antibodies (1:500) or with an anti- CaV2.1 IgG (1:10000) as a positive control. E) Immunoprecipitation experiments of induced (+) or non-induced (-) T-rex 293 cells using either ALS-IgGs or an anti-CaV2.1 antibody as a positive control. An aliquot of the original lysate was separated in the same gel to verify the correct induction of the CaV2.1 subunit (input). A negative control by omission of antibodies was also performed (beads). F) Synaptosomal proteins purified from mouse cerebellum (CaV2.1+/+, P17) subjected to immunoprecipitation experiments with ALS-IgGs samples. Analysis of precipitates was performed by western blotting using antibodies against several pre-synaptic proteins: synaptotagmin I (1:500), syntaxin 1A (1:1000), synaptophysin (1:400) and synaptobrevin II (1:500). A fraction of the initial lysate was run in parallel (input). Controls by omission of antibodies (beads) or immunoprecipitation of the pre-synaptic proteins themselves (PRE) were also included.
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