AnnNeurol—Chen Wanjin/Fan Dongsheng's research group revealed that heterozygous mutations in the SerRS gene cause peroneal muscular atrophy

Editor—Summer Leaf

Charcot-Marie-Toothdisease (CMT) is the most common inherited peripheral neuropathy and is typically clinically characterized by adolescent onset, slow-progressing muscle weakness and atrophy in the distal extremities with or without sensory disturbance [1].

。 It has been identified that there are as many as 100 CMT pathogenic genes, involving genes
encoding myelin structure, axon transport, cytoskeleton and other related proteins.
Although a variety of disease-causing genes have been cloned, 30~40% of CMT patients have not been diagnosed
with genes.
Moreover, most known genes are widely expressed in different tissues in the human body, and the mechanism by which mutations lead to specific damage to the peripheral nervous system is still unclear [2-4].

In the study of CMT mechanisms, the aminoacyl-tRNAsynthases (ARSs) family played an important role
.
To date, at least 6 ARSs heterozygous mutations have resulted in CMT, and most mutations are located in the aminoacylation functional domain of ARSs [5,6]
。 In CMT-associated ARSs mutations, defects in aminoacylation activity are not common, suggesting that simple loss of aminoacylation is not a prerequisite for the pathogenesis of CMT [7,8].

。 In addition, in animal models of CMT associated with ARSs, overexpression of wild-type ARSs did not mitigate peripheral nervous system lesions, but allele-specific RNA Disturbances can improve neuropathies, indicating that CMT-associated ARSs mutations are caused by dominant negative effects [9].

So, whether there are mutations in other ARS genes in undiagnosed CMT patients, and the role of ARS mutations in the pathogenesis of CMT remains unclear
。

Recently, Chen Wanjin's research group of the First Affiliated Hospital of Fujian Medical University, Fan Dongsheng's research group of Peking University Third Hospital and Henan Provincial People's Hospital cooperated in the Annalsof neurology A research paper titled "Heterozygous Seryl-tRNA synthetase 1 variants cause Charcot-Marie-Toothdisease" was published online A new aminoacyl tRNA synthase, serine tRNA synthase 1 (SerRs), was discovered as a new pathogenic gene for CMT, indicating a mutation SerRS protein causes decreased serine tRNA synthase activity and abnormal dimerization, which subsequently affects the rate of total protein synthesis and upregulation of eIF2α phosphorylation
.
(Extended reading: The latest research results of Fan Dongsheng's team can be found in the "Logical Neuroscience" report (click to read): Front Aging Neurosci - Fan Dongsheng team focus.
) Pathological role of NK cells in Parkinson's disease; Front Aging Neurosci Review—Fan Dongsheng's team focused on the interaction between peripheral and central immune system in amyotrophic lateral sclerosis).

Based on the previous stage, the First Affiliated Hospital of Fujian Medical University and the Third Hospital of Peking University established a standardized CMT clinical registration cohort (NCT04010188 and NCT04967716) and genetic testing process
.
Through detailed clinical data analysis, the researchers found that a total of 16 patients from 3 autosomal dominant and known gene-negative CMT families had high clinical phenotypic consistency
.
Using family chain analysis, it was found that the three CMT families shared the same chain interval
at 1p13.
3.
Further according to the genetic homogeneity model, linkage analysis combined with three families found that the common linkage interval was chr1:108681600-110300504 (combinedZmax LOD =6.
9), and found to be in this interval The SerRS gene had heterozygous mutations in all three families, all in the highly conserved aminoacylation functional domain, and all of them conformed to the co-separation phenomenon (Figure 1).

。

Figure 1.
Identification of heterozygous mutations in the SerRS gene in the CMT family

(Source: Jin He et al.
, Ann Neurol, 2022).

SerRS, as a kind of aminoacyl tRNA synthetase, is composed of three functional domains: tRNA binding functional domain, aminoacylation functional domain and UNE-S, and its main role is to bind amino acids to its corresponding tRNA , participate in the body's protein synthesis [6].

Notably, all CMT-associated ARS mutations function in vivo in the form of dimers
.
The researchers first demonstrated that heterozygous mutations in SerRS gene did not affect the expression
of the protein itself in patient-derived skin fibroblasts and 293T cells overexpressing the mutant protein.
In pyrophosphate release experiments, the researchers found that mutating the SerRS protein caused a decrease
in aminoacylation activity.
In 293T cells, mutant and wild-type SerRS proteins were overexpressed, and it was found that the mutant proteins could bind more wild-type proteins
through co-immunoprecipitation.
And in stochastic optical reconstruction microscopy (STORM) imaging, patient-derived fibroblast SerRS was found The volume of protein polymers increased
significantly compared with normal controls.
The above results show that the heterozygous mutation of the SerRS gene does not affect the expression of the protein itself, but reduces the activity of serine tRNA synthase and leads to abnormal dimerization (Figure 2).

。

Figure 2 The expression of the mutant SerRS protein was not affected, but it led to a decrease in aminoacylation activity and abnormal dimerization

(Source: Jin He et al.
, Ann Neurol, 2022).

SerRS is responsible for the aminoacylation of tRNA in the body, which in turn participates in the initial stage of protein synthesis, and its mutations affect the rate of synthesis of
new proteins.
Through puromycin insertion experiments, the investigators found that the rate of total protein synthesis of patient-derived skin fibroblasts decreased
.
For SerRS protein, which was overexpressed in 293T, the researchers found that only co-transfection of wild-type and mutant SerRS led to a significant decrease in the rate of total cell protein synthesis (Fig 3）
。

Figure 3 Mutation of the SerRS protein causes a decrease in the rate of total protein synthesis in cells

(Source: Jin He et al.
, Ann Neurol, 2022).

In mouse and Drosophila models associated with ARSs mutations, ARSs mutations led to abnormal protein synthesis leading to ribosome arrest, phosphorylation of eIF2α and activation of the integrated stress response ().
Integrated stress response (ISR), further affecting neuronal translation profile changes [10].

The researchers first demonstrated upregulation
of phosphorylated eIF2α in patient-derived skin fibroblasts.
In 293T cells that overexpressed mutant proteins, the researchers also found that only co-translating wild and mutant types could lead to significant changes in phosphorylated eIF2α, and verified the same phenomenon
in the neuronal cell line N2a.
Together, these experiments show that mutated SerRS is involved in the pathogenesis of CMT through dominant negative effects (Figure 4).

。

Figure 4 Mutation of the SerRS protein leads to an increase in phosphorylated eIF2α levels

(Source: JinHe etal.
, AnnNeurol, 2022).

In summary, the study was based on three autosomal dominant CMT families, and a new CMT pathogenic gene SerRS was identified through family linkage, and it was found that heterozygous mutations in SerRS gene led to a decrease in aminoacyl capacity and abnormal
protein dimerization.
In addition, further testing of puromycin insertion experiments and eIF2α phosphorylation levels revealed that CMT caused by mutations in SerRS may be mediated
by overt negative effects.
This study can not only provide diagnostic basis and genetic counseling for more undiagnosed CMT patients, but also further verify the role of protein synthesis and phosphorylation eIF2α in the pathogenesis of CMT, laying a foundation
for subsequent exploration of pathogenesis and therapeutic targets.
The disadvantage is that the study only explored the relevant molecular changes at the cellular level, and subsequent animal experiments are still needed to further verify the research conclusions and explore the mechanism
of peripheral nerve damage caused by abnormal protein synthesis.