Autism-Linked Mutations in α₂δ-1 and α₂δ-3 Reduce Protein Membrane Expression but Affect Neither Calcium Channels nor Trans-Synaptic Signaling

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Although risk genes have been reported in autism patients, their functional relevance to the development of the disease is poorly understood. The authors investigated two mutants of the α2δ family and found that these mutations affected the membrane expression and synaptic localization without affecting the current properties or trans-synaptic signaling. How they contribute to the pathogenesis of ASD still remains to be further studied.

There are a few suggestions.

1.       Live cell imaging revealed reduced membrane localization of the α2δ mutants compared the WT counterparts, and Westering blotting showed that the overall expression levels were not changed. Is it possible to also utilize whole cell fluorescence to quantify the overall expression level to cross-validate the Western blotting results?

2.       It would be also more reassuring to demonstrate the α2δ expression levels and patterns in mouse hippocampal neurons.

3.       Quality of the Western blotting figures need to be improved. The bands are faint and pixelized.

4.       In Figure 6B left panel, there was a trend that the R351T mutant transfected cells showed less prominent increase in current amplitude compared to the WT. Could the difference be better detected when the cells are stimulated? Overall, the current study only investigated the non-stimulated cells. It would be quite interesting and informative to study the effects of the mutant on neurotransmission properties under certain stimulation conditions.

Author Response

Point-by-point response to the reviewer comments:

Response to referee 1:

Although risk genes have been reported in autism patients, their functional relevance to the development of the disease is poorly understood. The authors investigated two mutants of the α₂δ family and found that these mutations affected the membrane expression and synaptic localization without affecting the current properties or trans-synaptic signaling. How they contribute to the pathogenesis of ASD still remains to be further studied.

There are a few suggestions.

1. Live cell imaging revealed reduced membrane localization of the α₂δ mutants compared the WT counterparts, and Westering blotting showed that the overall expression levels were not changed. Is it possible to also utilize whole cell fluorescence to quantify the overall expression level to cross-validate the Western blotting results?

• In parallel to the live cell staining shown and analyzed in figure 4 we routinely stained fixed/permeabilized cells to rule out differences caused by eventual effects on overall expression levels. These staining were qualitatively checked in parallel to each live cell staining experiment. They repeatedly showed no apparent difference in overall expression levels. Because we used the WB experiments for quantification, we decided at the time to not acquire large sets of images for quantifying expression levels in fixed/permeabilized cells. A typical example of such parallel staining is shown below in a figure (Fig. R1) intended for the review process only. The reviewer also suggested performing analysis of the overall expression levels in neurons (see below), which we have thoroughly performed on images acquired in parallel with the neuronal live cell staining experiments (see below) and which we have now added to the results (see below and new figure 3). Because this analysis also did not reveal any indication for differences, we feel that repeating the experiments in tsA201 cells to further prove this obvious observation is not warranted.

Figure R1. The p.R351T mutation does not compromise the total expression level of α₂δ-1 in tsA201 cells. Representative images of anti-HA permeabilized-labelled tsA201 cells co-transfected with soluble eGFP together with either WT or mutated α₂δ-1 (R351T), both of which are tagged with double HA tag at the N-terminus. Scale bars, 20 μm.

2. It would be also more reassuring to demonstrate the α₂δ expression levels and patterns in mouse hippocampal neurons.

• In contrast to the tsA201 cell experiments (see above) we have indeed recorded a large number of images of fixed/permeabilized hippocampal neurons. These experiments were performed in parallel to the anti-HA live cell experiments of hippocampal neurons presented in figure 2. We have now performed a full analysis of total wildtype and mutant α₂δ-1 and α₂δ-3 expression levels based on the anti-HA labeling of fixed/permeabilized hippocampal neurons. These data have now been added to the manuscript as new figure 3. Details on the quantification procedures were added to the respective methods section (see page 22f). These experiments confirm that overall protein expression levels in neurons are not changed, and hence strongly support our initial conclusion that the mutations specifically affect neuronal surface expression. We thank the reviewer for this helpful suggestion.

3. Quality of the Western blotting figures need to be improved. The bands are faint and pixelized.

• We thank the reviewer for pointing out this quality issued. We think that it was largely due to the image compression used for the PDF file. However, to rule out any quality loss based on the compression algorithm we have improved the print quality of the original WB images. Regarding the faintness of the bands, we now used images recorded with a longer exposure time. This worked well for the presentation of most of the bands, however, it also leads to a slight saturation effects of two bands presented in figure 5.

4. In Figure 6B left panel, there was a trend that the R351T mutant transfected cells showed less prominent increase in current amplitude compared to the WT. Could the difference be better detected when the cells are stimulated? Overall, the current study only investigated the non-stimulated cells. It would be quite interesting and informative to study the effects of the mutant on neurotransmission properties under certain stimulation conditions.

• To study the effects of α₂δ mutations on the channels we performed electrophysiological analysis in tsA201 cells. This method is the current state-of-the-art approach to characterize the biophysical properties of calcium currents (Haddad et al., 2024, Nikonishyna et al., 2022, Ortner et al., 2017). To this end we co-transfected tsA201 cells with the subunits of voltage-gated calcium channels (α₁, β, with or without α₂δ) together with soluble eGFP to identify positively transfected cells. 48 hours after transfection, calcium currents passing through calcium channels expressed at the cell membrane were measured in GFP-positive cells in whole-cell configuration and voltage-clamp mode. Cells were stimulated sequentially to different 50-ms-long test potentials in 5 mV voltage-steps from -80 to +80 mV. Calcium currents were recorded during each stimulation step. Like the reviewer, we also initially suspected a partial effect based on the less prominent increase in current density, albeit not statistically significant (p=0.35, ANOVA followed by post hoc Tukey analysis). Therefore, and in addition to Ca_V3, we decided to also study the effect of R351T on another L-type calcium channel, the Ca_V1.2 isoform. This analysis revealed a near-identical IV-curve for R351T and WT α₂δ-1 (Fig. 8). Moreover, in a completely independent set of experiments, which is part of another study and hence not included in the present manuscript, we performed basically the same experiment with Ca_V1.3, but with another negative control (an α₂δ-1_ΔMIDAS mutant). In this set of experiments, the IV curves of channels with α₂δ-1_R351T vs. α₂δ-1 WT were basically indistinguishable (see Fig. R2, which is shown only for the review process). This confirms that the mild differences seen in figure 7 are indeed within the expected statistical variability. 

Figure R2. α₂δ-1_R351T increases current densities of Ca_V1.3 channels similar to WT α₂δ-1. Current-voltage relationships of Ca_V1.3 channel recorded from tsA201 cells co-transfected with Ca_V1.3 and β₃ together with α₂δ-1_ΔMIDAS (grey triangles), or with α₂δ-1 (blue circles), or α₂δ-1_R351T (red rectangles). Recordings were obtained from three independent experiments.

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

In this paper the authors demonstrate the effects of two mutations found in α2δ calcium channel subunits whose mutations (α2δ-1 p.R351T and α2δ-3 p.A275T) are apparently related with the autism spectrum disorder (ASD). This is an elegant work with several molecular and functional experiments that demonstrate most of their hypothesis. Unfortunately, the main weakness of the work is the heterologous expression system used for most of the experiments despite having a homologous expression system (much more interesting and physiologically relevant). For this reason, due to the possible mainly functional consequences of performing these experiments in a neuronal environment, I strongly recommend to the authors performing a molecular and functional characterization of such mutations in the homologous expression system that will allow them to definitively make their conclusions in a much more relevant system. In addition, is necessary to complete the molecular characterization of Figure 3 in the α2δ-3 p.A275T mutation and not only in the R351T.

Author Response

Point-by-point response to the reviewer comments:

Response to referee 2

Dear Authors,

In this paper the authors demonstrate the effects of two mutations found in α₂δ calcium channel subunits whose mutations (α₂δ-1 p.R351T and α₂δ-3 p.A275T) are apparently related with the autism spectrum disorder (ASD). This is an elegant work with several molecular and functional experiments that demonstrate most of their hypothesis. Unfortunately, the main weakness of the work is the heterologous expression system used for most of the experiments despite having a homologous expression system (much more interesting and physiologically relevant). For this reason, due to the possible mainly functional consequences of performing these experiments in a neuronal environment, I strongly recommend to the authors performing a molecular and functional characterization of such mutations in the homologous expression system that will allow them to definitively make their conclusions in a much more relevant system. In addition, is necessary to complete the molecular characterization of Figure 3 in the α₂δ-3 p.A275T mutation and not only in the R351T.

• We appreciate that the reviewer considers our study elegant and of high quality. We fully agree with the reviewer (and also mention this repeatedly in our manuscript, e.g., abstract and discussion) that ultimately α₂δ protein disease mutations need to be investigated in their native physiological environment. In this regard and based on our experience the most powerful model system is provided by differentiated hippocampal neurons cultured at low density. These neurons express substantial amounts of three different α₂δ protein isoforms (α₂δ-1, α₂δ-2, and α₂δ-3; see (Schlick et al., 2010)). As we and others have previously shown, α₂δ isoforms act partially redundant as auxiliary calcium channel subunits on the one hand (reviewed in (Dolphin, 2012, Dolphin, 2016)), and as synaptic organizers on the other hand (Ablinger et al., 2022, Schopf et al., 2021). Hence, wild-type hippocampal neurons cannot simply be used as homologous expression models, because the effects of α₂δ loss-of-function mutations will be masked by the redundant actions of the endogenous α₂δ Neuron-like cell lines do not qualify as alternative homologous expression models, because their neuronal differentiation is only rudimentary and often not based on physiological mechanisms. Taken together, we are fully aware of the need of a powerful native homologous expression system. For exactly that reason our laboratory has, over the last two decades, strongly contributed to the development and implementation of novel homologous expression models (Ablinger et al., 2022, Geisler et al., 2019, Haddad et al., 2024, Obermair et al., 2005, Obermair et al., 2008, Schopf et al., 2021, Tuluc et al., 2007). Due to the complex endogenous expression patterns of α₂δ proteins, however, homologous expression of mutated α₂δ proteins requires a null background of specific α₂δ isoforms. To this end and because existing α₂δ knock-out mouse models suffer from severe neurological disorders and associated difficulties in breeding regimes (Geisler et al., 2021, Schopf et al., 2021), we are currently developing specific conditional in vitro model systems allowing the future homologous expression and investigation of individual α₂δ protein isoforms and mutations. Developing such model systems, however, requires many years of work, patience, and perseverance. Therefore, until more sophisticated homologous expression models become available, the combination of established state-of-the-art heterologous expression systems (see also (Haddad et al., 2024, Nikonishyna et al., 2022, Ortner et al., 2017)) with homologous expression for studying membrane trafficking, localization, and trans-synaptic signaling provides a powerful experimental approach.
• Regarding the comment that “[it] is necessary to complete the molecular characterization of Figure 3 in the α₂δ-3 p.A275T mutation and not only in the R351T”: A275T mutation, which resulted in only a minor reduction in α₂δ-3 membrane expression, the R351T mutation drastically decreased the membrane expression of α₂δ-1 to more than 50% of WT protein (confer Fig. 2). Because the functional electrophysiological analysis was done in tsA201 cells we therefore decided to also analyze cell surface expression of α₂δ-1_R351T also in these cells. Expression of α₂δ-3_A275T in tsA201 cells neither altered calcium current, nor did it affect total proteins expression (see Fig. 5). Therefore, we are convinced that an additional analysis of the cell surface expression of α₂δ-3_A275T in tsA201 cells will not change the conclusions reached in our present study and decided to not perform these additional experiments.

 

References

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Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I have no additional comments.