Amyotrophic lateral sclerosis (ALS) is the commonest motor neuron disease (MND), which is characterized by the damage of both upper and lower motor neurons, manifested as the progressive weakness, muscular atrophy and fascicular fibrillation of muscle as well as pyramidal tract signs. Most patients died of respiratory paralysis within 3–5 years [1]. Current clinical used drugs only could partly postpone the disease progression of ALS, their effect mechanisms include the elimination of oxygen free radicals and the protection of neurons. Up to now, there haven’t been any reliable specific drugs curing ALS. Several studies have found that the pathogenesis of ALS is closely related to genetic factors, the toxicity of excitatory amino acid, the damage of oxidative stress, the damage of axonal transport, the defect of mitochondrial function, and the abnormal aggregation of protein so on. Their effects can exert synergistic effects each other [2]. Recently found pathogenic mechanisms of ALS include RNA metabolic dysfunction, the defects of cytoplasmic transport, and the dynamics damage of RNA particles and other ribonucleoprotein body [1]. These pathogenic factors can lead to the disorder of gene transcription, splicing, translation and other related processes, and further damage cells and /or tissues.
The pathological feature of ALS is the generation of cytoplasmic inclusion bodies, aggregating in damaged motor neurons and oligodendrocytes. Inclusion bodies not only exist in spinal cord, but also exist in multiple brain regions, such as frontal cortex, temporal cortex, hippocampus and cerebellum [3, 4]. The aggregation of abnormal proteins and the generation of inclusion bodies have been considered as the pathological characteristics of several neurodegenerative diseases including ALS. Among them, the aggregation of abnormal proteins and the generation of inclusion bodies are the commonest in spinal motor neurons. Ubiquitin positive inclusion body is a sign of ALS pathology. In ALS, some proteins encoded by mutant genes, such as SOD1, TDP-43, FUS, OPTN, Ubqln2 and NEFH, are the important components of inclusion bodies [5–7].
In 1993, the first mutant SOD1 gene [8] was found in the patient of familial ALS (fALS). From then on, more than 50 potential pathogenic genes of ALS have been found up to now, among them, including c9orf72, TDP-43, FUS, heterologous ribonucleoprotein (hnRNP) A1, Sqstm1, VCP, OPTN, Pfn1 [9]. These genes are roughly divided into the following categories: The genes affected the control of protein stability and expression quality, the genes affected the normal function and metabolism of RNA, and the genes interfered with the cytoskeleton dynamics of distal axons of motor neurons. The advances in studied technologies have prompted researchers to sequence a wide range of DNA in patients with sporadic ALS (sALS), and found that the genetic variation of ALS gene isn’t uncommon. 1–3% of sALS patients are caused by the missense mutations in SOD1 gene [10], and 5% or more sALS are caused by the abnormal amplification of introns in c9orf72 gene [11].
In 2006, Lee et al. found that RNA binding protein (RBP) TDP-43 deposited in brain and spinal cord of ALS patient, and aggregated in cytoplasm. TDP-43 mislocation is now widely recognized as the characteristic of partial sALS and fALS [12]. Subsequently, the mutations of other ALS related proteins including FUS and hnRNP A1 were identified. These proteins were mutated after bound with RNA, which made RNA biological role be more concerned in the pathogenesis of ALS. Because the mutations of RNA binding proteins (RBPs) gene are found in neurodegenerative diseases, the dysfunction of RBPs is clearly evolving into the central theme of neurodegenerative diseases, and the dysfunction of RNA processing and the phagocytosis of aggregation protein become an important mechanism related to ALS [13]. The RBPs closely related to ALS include TDP-43, FUS, c9orf72, hnRNP A1, hnRNP A2/B1, Matr3, SETX, ELP3, atxn2, ANG, SMN1 and SMN2 [14, 15]. In addition, many other RBPs have been found to show changes in subcellular distribution in the neurons and/or glial cells of ALS patients, but the mutations caused ALS have not been known [16], which indicates that even if the gene of RBPs does not mutate, it can also cause the destruction of RNA stability in ALS, or affect the metabolism and stability of RNA during the occurrence and development of ALS. Only 4% of fALS patients show the gene mutation of TDP-43, about 97% of ALS patients had dis-localization and the inclusion body of TDP-43 protein. It suggests that even if there is no relevant mutation, the cytoplasmic and intranuclear inclusion bodies are common in ALS patients [17].
Both TDP-43 and FUS are involved in RNA related pathways, they play some roles at many steps of RNA regulation, including RNA transcription, splicing, transportation, translation and microRNA production [18]. Both TDP-43 and FUS proteins interact directly with polyphase ribonucleoprotein complexes, regulate RNA splicing and transport, both TDP-43 and FUS have the similar biological roles in RNA related pathways [19]. The cellular cytotoxicity of mutants and/or cytoplasmic dislocated TDP-43 and FUS might be caused by the following reasons: (1) the loss of normal nuclear function leads to the disorder of nuclear RNA processing; (2) obtains the additional cytoplasmic RNA binding activity; (3) the polymerization dependent toxicity [7, 20–22]. More and more evidences show that the neurotoxicity generates through various cellular pathways, such as the RNA mismatch and the transcription reduction of c9orf72 gene, the disorder of transport between nuclear and cytoplasm, the stress of nuclear protein and DNA damage [23–27]. The break of DNA strand is the most serious types of DNA damage. If the broken DNA are not repaired properly, which usually lead to cell death. The hnRNP A1 is a member of hnRNP family, involves in a variety of RNA metabolism [28]. Several studies find that the hnRNP A1 negatively regulates its mRNA expression by inhibiting the intron 10 splicing of hnRNP A1 pre-mRNA in cell models. This mechanism might be the self-regulation of hnRNP A1 expression, and the low-level hnRNP A1 overexpression can cause cytotoxicity by activating the pathway of mitochondrial apoptosis [29, 30]. It is suggested that the level of hnRNP A1 is strictly controlled within a certain range by the self-regulation of mRNA, so that no cytotoxicity of hnRNP A1 expression occurs at the physiological condition.
Among recent discovered RBPs, hnRNP G is the most prominent in the familial members of more than 20 RBPs proteins. TDP-43, FUS and hnRNP A1 are members of hnRNP protein family that regulate RNA metabolism at each stage of RNA life cycle [31–33]. hnRNPs help to control the maturation of newly formed hnRNA and pre-mRNA, stabilize mRNA and control its translation during cell transport. hnRNPs are key proteins in cellular nucleic acid metabolism [34]. Since neurons are the permanent cells, the steady state of mRNA needs to be strictly regulated, and the steady state of mRNA is very vulnerable to the dysfunction of RBPs including hnRNPs [35].
hnRNP G is the product of RNA binding motif protein and RNA binding motif protein X-linked (RBMX) gene, is the only glycosylated hnRNP that can directly bind to RNA. The RNA binding motif protein Y-linked gene is located on Y chromosome, is formed by the reverse transposition of RBMX [36–38]. hnRNP G can be used as a pre-splicing factor of mRNA, involves in the selection of specific splicing sites, is related to the post transcriptional new mRNA of RNA polymerase II, and regulates the selection of pre-mRNA alternative splicing sites [39]. Recent studies have shown that hnRNP G is an active regulator of splicing mechanism in neurodegenerative diseases. Firstly, hnRNP G promotes the expression of exon 7 of survival motor neuron. The homozygous deletion of hnRNP G gene leads to the common motor neuron disease called the spinal muscular atrophy [40]. Secondly, the microtubule associated protein like Tau is regulated by the interaction of cis- and trans- factors including hnRNP G. In human genes, the exon 10 of Tau is a binding domain of selective splicing, the wrong splicing of Tau gene results in the frontotemporal dementia of Parkinson's syndrome [41]. It is obvious that hnRNP G has various and complex functions. Our studied groups found that the hnRNP G protein in the spinal cord of TG(SOD1*G93A)1Gur (TG) mice was significantly reduced through the methods of proteomic analysis [27]. The purpose of this study is to further explore the possible roles and mechanisms of hnRNP G in the pathogenesis of ALS.