Population characteristics
The study population include two male groups: 13 FX subjects with a mean age of 28,4 ± 4,6 years and 14 healthy controls, with a mean age of 30,8 ± 7,8 years. Individual characteristics of each participant, including age, FX diagnostic and medication are listed in Additional file 2.
Platelets 35S incorporation rate is too low for efficient protein synthesis measurement
The methodology used in this study was based on radiolabelled amino acids incorporation method to ensure an unbiased and robust measurement of proteins synthesis [33]. Using this method, we first investigated the relevance of using blood platelets for the monitoring of protein synthesis rate alterations in FXS. Indeed, platelets are one of the most valuable peripheral model for FX due to their close biochemical similarities with neuron and the fact that they replicate some of FXS core molecular alterations [31]. However, the translational machinery of platelets is scarce and devoted to the production of a limited number of proteins during its short lifespan of 10 to 14 days [35]. As expected, even after optimization of the method, the rate of protein synthesis is barely measurable in platelets (Fig. 2A-B) making them unsuitable for our purpose.
We therefore decided to use PBMCs, which also replicate some of FX core biochemical and molecular defects, as a model to monitor protein metabolism alteration in FXS. Following adjustment for protein content of cells, protein synthesis rate measured in PBMCs is 3 thousand-fold higher than in platelets (Fig. 2A-B). This observation clearly demonstrates that PBMC’s rate of protein synthesis is sufficiently high to assess differences between FXS and control population.
Rate of protein synthesis is decrease in fragile X PBMCs
One of the greatest challenge encounters when undergoing ex vivo experiments on human peripheral cells is the individual heterogeneity inherent to the use of such models. The sources of this interindividual variability are highly diverse (genetic background, epigenetic factor, life habits, drugs consumption, etc.) and can easily concealed differences between control and affected cells. A stringent and robust experimental protocol is thus mandatory to limit those fluctuations and ensure results reliability. In concordance with this mindset, we firstly ascertain our assay optimization by investigating intraindividual variability. As illustrated in Fig. 3A, our method shows an intraindividual variability inferior to 10%, even when measurements were made several months apart (Table 1). This great reproducibility will obviously facilitate our ability to detect small difference regarding rate of protein synthesis between FX and CTL PBMCs. Therefore, we confidently measured rates of protein synthesis from PBMCs obtained from 13 FX males and 14 matched control individuals. The average rate of protein synthesis was 1830 ± 141 cpm/min for the FX group as compared to 2500 ± 194 cpm/min for the control group (Fig. 3B). This represent a 26.9% decrease of PBMCs rate of protein synthesis in the FX cohort (p = 0.0193). No correlation was found between subject age and rate of protein synthesis in both groups (Additional file 3).
Table 1: Rate of protein synthesis measurement reproductibility
(Should be place with or after Figure 3)
|
|
|
|
|
ID
|
Mean rate of protein synthesis measurement (cpm/min)
|
Absolute Difference between replicate
|
Variation (Difference/Mean)
|
Days elapsed between replicate
|
Fragile X patients
|
X1
|
2209
|
62
|
2.8%
|
175
|
X4
|
1438
|
326
|
22.7%
|
35
|
X8
|
1646
|
137
|
8.3%
|
70
|
X10
|
1734
|
69
|
4.0%
|
63
|
X13
|
1453
|
99
|
6.8%
|
29
|
Control individuals
|
C1
|
2469
|
66
|
2.7%
|
4 and 107
|
C2
|
2221
|
40
|
1.8%
|
214
|
C3
|
2632
|
194
|
7.4%
|
131
|
C4
|
3270
|
125
|
3.8%
|
126
|
C5
|
1356
|
268
|
19.7%
|
164
|
C6
|
2897
|
26
|
0.9%
|
125
|
C7
|
2111
|
374
|
17.7%
|
155
|
C10
|
1684
|
231
|
13.7%
|
9
|
To our knowledge, our results constitute the very first report of a decreased in the rate of protein synthesis in a human peripheral cellular model of FX. As such, the decreased protein synthesis rate measured in FX PBMCs is in concordance with the only two reports to ever addressed that topic in FX neurons. Indeed, the Beebe Smith group used PET imaging to measure a decreased in [11C] leucine integration into nascent brain proteins of FX individuals in two independent studies. This fact, in addition to the great reproducibility shown by our method, clearly demonstrates the relevance of using PBMCs as a model to investigate translational alterations in FXS.
Our observations are different to those reported in actively dividing cells, namely FX fibroblasts, where an increase in protein synthesis was observed. The translational machinery in such cells is much more active and focused on sustaining the resource-intensive processes of cellular division whereas in non-proliferative cells, protein synthesis is limited and mainly dedicated to cell maintenance. It is therefore conceivable that the absence of FMRP may have opposite effect in cells having contrasting translational processes. In this regard, we believe that the alterations in the rate of protein synthesis found in FX PBMCs may display with more fidelity the defect found in neurons, which are also terminally differentiated cells. The adverse trend in protein synthesis defects between PBMCs and fibroblasts may also be explained by divergence regarding FMRP function between those two models.
Curiously, the two fibroblasts-based studies report poor reproducibility of the measurements made exclusively in FXS cell lines. Human skin fibroblasts gradually loose their potency to divide during cell passages and protein synthesis rate is affected along the way. The reported large differences in protein synthesis rate in FX fibroblasts replicates could be the signature of distinct effect of FMRP in protein synthesis whether the potency to divide of the cells is still high or at time they get into senescence. Alternatively, the culture conditions used in those two studies may have also influence the translational activity by inducing a metabolic stress on the fibroblasts, a phenomenon that can impact rate of protein synthesis measurement in a FMRP-dependant manner [36].
For those reasons, we believe that the use of a freshly extracted and non-proliferative model will give a better reflection of the protein metabolism found in neuron, which are primary and terminally differentiated cells, than immortalised or long-lasting cultured cellular models.
Assessment of PBMCs rate of protein synthesis as a biomarker for FXS
In order to ascertain the pathophysiological role of protein synthesis in FXS, we evaluated the association between rate of protein synthesis and the clinical phenotype of FX individuals Unfortunately, such correlations were not observed for the cognitive evaluations assessed in this study (Fig. 4,Table 2 and additional file 4). Similar results were obtain with a larger cohort of fibroblasts derived from 21 FX males [37].
Table 2: Association between rate of protein synthesis measurements and cognitive evaluations
(Should be place after or with Figure 4)
Variable
|
Pearson correlation
|
Spearman correlation
|
r
|
p
|
r
|
p
|
ABC-C score
|
-0.195
|
0.616
|
-0.250
|
0.521
|
Full Scale IQ
|
0.239
|
0.569
|
0.156
|
0.716
|
Brief score
|
0.234
|
0.545
|
0.251
|
0.511
|
ABAS II score
|
-0.250
|
0.517
|
-0.293
|
0.442
|
This lack of association in our study can easily be explained by the study design. Indeed, we purposely recruited fully mutated FX males in order to restrain the heterogeneity of the FX cohort enrolled.; the downside being that all FX subjects presents low cognitive profile. Our strategy, combined with a highly reproducible methodology, allowed us to monitor a significant difference between the FX and control group with a relatively small sample size. On the other hand, this approach did not allow us to get a comprehensive portrait of the translational defect in a sample representative of the entire FX population. As such, our data cannot rule out if the amplitude of the rate of protein synthesis alteration found in FX individuals is related to the severity of the clinical presentation; such observations being out of the scope of the present study.
Clinical trials of FXS are currently limited by the lack of objective tools to assess treatment efficiency in individuals. The development of such monitoring biomarkers could drastically improve the scope of those studies and promotes a better understanding of the human physiopathology [38]. As seen in animal researches, rate of protein synthesis normalisation could be used to address this issue [18, 19]. We truly believe that the method described in this paper is well suited for this task. Indeed, our radiolabeled assay showed great intraindividual reproducibility and robustness, which mean that it could easily detect improvement in rate of protein synthesis upon treatment. Furthermore, unlike fibroblasts, PBMCs are a well designated model for human trials due to their non-invasive nature, ease of collection and the fact that they replicated the protein synthesis defect found in FX neurons [22, 23]. Nonetheless, the utilisation of this procedure in large multi-center trial might be somewhat limited due to the fact that it must be done in a timely fashion.