A motor neuron strategy to save time and energy in neurodegeneration: adaptive protein stoichiometry

Abstract Neurofilament proteins (Nf) are a biomarker of disease progression in amyotrophic lateral sclerosis (ALS). This study investigated whether there are major differences in expression from in vivo measurements of neurofilament isoforms, from the light chain, NfL (68 kDa), compared with larger proteins, the medium chain (NfM, 150 kDa) and the heavy (NfH, 200‐210 kDa) chains in ALS patients and healthy controls. New immunological methods were combined with Nf subunit stoichiometry calculations and Monte Carlo simulations of a coarse‐grained Nf brush model. Based on a physiological Nf subunit stoichiometry of 7 : 3 : 2 (NfL:NfM:NfH), we found an ‘adaptive’ Nf subunit stoichiometry of 24 : 2.4 : 1.6 in ALS. Adaptive Nf stoichiometry preserved NfL gyration radius in the Nf brush model. The energy and time requirements for Nf translation were 56 ± 27k ATP (5.6 h) in control subjects compared to 123 ± 102k (12.3 h) in ALS with ‘adaptive’ (24:2.4:1.6) Nf stoichiometry (not significant) and increased significantly to 355 ± 330k (35.5 h) with ‘luxury’ (7:3:2) Nf subunit stoichiometry (p < 0.0001 for each comparison). Longitudinal disease progression‐related energy consumption was highest with a ‘luxury’ (7:3:2) Nf stoichiometry. Therefore, an energy and time‐saving option for motor neurons is to shift protein expression from larger to smaller (cheaper) subunits, at little or no costs on a protein structural level, to compensate for increased energy demands.

the plates were rinsed 3 times with 200 μl of wash buffer as before. Then 25 μl of Heteroblock (Omega Biologicals) were added to the standard and blank well at a concentration of 300 μg/ml. To the sample wells 25 μl of Heteroblock were added at a concentration of 600 μg/ml. To the prepared plate 25 μl of standard and sample were added in duplicates. The wells were sealed and incubated on the shaker for 2 hours at RT.
Following this, plates were washed as before 3 times. Next, the detecting mouse biotinylated detector antibody (2:1, UmanDiagnostic, Sweden) was diluted in TBS containing 1% Bovine Serum Albumin, 0.1% Tween 20, pH 7.5. Of this 25 μl were added to each well. Plates were sealed and incubated on the plate shaker at RT for 1 hour. After a final washing cycle (3x), 25 μl of SULFO-TAG TM labelled streptavidin (MSD, Gaithersburg, USA) was added to 150 μl of read buffer (MSD, Gaithersburg, USA) to generate electrochemiluminescence and the signals were detected with a SECTOR Imager 2400 camera (MSD, Gaithersburg, USA). The quality control sample was used to record the inter-assay variation and for additional one-point calibration as described [Petzold et al. 2010].

Neurofilament medium chain (NfM)
We have used a commercially available ELISA kit (Cloud Clone Corp., product no. SEB326Hu) to measure plasma NfM, based on a sandwich ELISA with two different polyclonal antibodies against NfM. Antibodies, protein standards as well as detection and dilution reagents were all provided by the kit; standard, detection reagent A and B and the 96-well strip plate have been kept stored at -20° C upon receipt till use within one month. Standard curve was reconstituted by diluting Standard (NfM provided by the kit at a concentration of 4000 pg/mL) to the highest standard fixed at a concentration of 1000 pg/mL, then performing a double dilution curve till the concentration of 31.2 pg/mL. No previous evaluation of pre-analytical variables in a complex matrix as plasma was available for NfM. We therefore assessed NfM levels after serial plasma dilutions with 0.01 mol/L phosphate buffered saline (PBS, as recommended by the manufacturer) and found a hook effect similar to NfH (Lu et al., 2011) (data not presented). In order to overcome this effect, we tested different conditions as described elsewhere (Lu et al., 2011) to achieve parallelism. For NfM we found that parallelism was achieved with a dilution factor of more than 1:8. Therefore, plasma samples were processed at 1:10 with 0.01 mol/L PBS. If not taken into account the hook effect can cause underestimation of the correct Nf concentration.
After having plated 100 µl of the standard, blank, and samples in duplicate in each well, the plate was incubated for one hour at 37°C on the shaker. Next, the liquid was removed and 100 µl detection reagent A, provided by the manufacturer, was added to each well, leaving the plate in incubation for one hour at 37°C.
Liquid was consequently removed again, and 350 µl wash solution were added to each plate by a multichannel pipette, let it rest a couple of minutes, and then removed again; this step was performed thrice.
After the last wash, 100 µl detection reagent B were added to each well and the plate was incubated for 30 minutes at 37°C. Next, the washing process was repeated 5 times as described above. After the last wash, 90 µl Substrate Solution were added to each well, and the plate was incubated for 15 minutes at 37°C with light shedding. Finally, 50 µl of Stop Solution were added to each well and, after gentle mixing, the plate was run on Microplate Reader at 450 nm. %. The quality control sample was chosen from a plasma sample with high NfM concentration. The quality control sample was used to record the inter-assay variation and for additional one-point calibration as ALREADY described [Petzold et al. 2010]. Intra-assay coefficient of variation (CV) was always below 20%, with an average value at 6.91%, while inter-assay CV was found at 2.94%. The detection curve of the kit was fitted between 15.1 and 1,000 pg/mL, with an overall analytical sensitivity above 6.1 pg/mL.

Neurofilament heavy chain (NfH)
Regarding NfH, an in-house ELISA for hyper-phosphorylated NfH (NfH SMI34 ), variably phosphorylated NfH (NfH SMI35 ) and for the sum of the two were employed as reported in Lu et al., 2015a. The two above mentioned capture antibodies were mouse monoclonal anti-NfH antibodies (Covance, USA) while the detector antibody was rabbit polyclonal anti-Neurofilament 200 (N4142; Sigma, UK). The reporter antibody was horseradish peroxidase (HRP)-labelled swine polyclonal anti-rabbit antibody (P0217; DAKO, Denmark). For the rest of the manuscript we will therefore refer to the sum of the effect of SMI34 and SMI35 as NfH. As for NfL, extensive analytical refinement was undertaken to assess analytical sensitivity, lower detection levels and linearity (Lu et al., 2012). This work included the study of the reported hook effect when testing linearity of NfH expression in dilution experiment with a standard protein. Pre-analytical treatment with urea was employed to overcome the hook effect which results from a lack of parallelism due to endogenous binding to heterogenous aggregates (protein fragments and IgG) (Lu et al., 2011). The quality control sample was used to record the inter-assay variation and for additional one-point calibration as described [Petzold et al. 2010]. The assay protocol is summarised in the Supplementary table 1.

Subgroup analyses for disease progression
To assess whether clinical variables used to stratify our ALS patients may introduce a bias and affect neurofilament molar ratio calculations, we tested if any statistically significant difference was detectable in ALS subsets at the extremes of progression rate (i.e. fast progressor and slow progressors). Supplementary table 1 shows that there is no significant difference between fast and slow progressors in terms of age at onset and sampling, smoking habits or cognitive involvement. This implies there is no potential covariate for influencing change in stoichiometry between these two sub-groups of patients.
The progression rate at time of Nf isoform sampling, baseline, was significantly related to NfM (GLM, p=0.03), NfH (GLM, p=0.03), but not NfL (GLM, p=0.13) concentrations (Supplementary table 2). The Bonferroni corrected p-value for the 3 progression groups calculates to 0.016. According to the Bonferroni corrections the post-hoc analyses revealed significantly higher NfM levels in fast progressors if compared to slow progressors (p=0.0074); as were NfH levels (p=0.0074).