Alzheimer’s Disease-Associated SNP Rs708727 in SLC41A1 May Increase Risk For Parkinson’s Disease: Report From Enlarged Slovak Study Part 1

Abstract:

SLC41A1 (A1) SNPs rs11240569 and rs823156 are associated with altered risk for Parkinson’s disease (PD), predominantly in Asian populations, and rs708727 has been linked to Alzheimer’s disease (AD). In this study, we have examined a potential association of the three aforementioned SNPs and of rs9438393, rs56152218, and rs61822602 (all three lying in the A1 promoter region) with PD in the Slovak population. 

Parkinson's disease is a neurological disorder that often affects people's ability to move their bodies, but many people may not know that Parkinson's disease can also affect people's memory. While this may worry some people, there are things we can do to improve our memory and maintain our physical and mental health.

First, understand the relationship between Parkinson's disease and memory. Parkinson's disease primarily destroys nervous tissue, especially dopamine neurons, which are important for the body's motor coordination. At the same time, dopamine neurons also affect our cognitive abilities, including attention, working memory, and executive function. Therefore, in the early stage of Parkinson's disease development, there may be symptoms of memory loss, which will gradually worsen.

Second, we should understand how to maintain a good memory. Although Parkinson's disease can be worrisome, there are ways we can help maintain a good memory. For example, measures such as good sleep, a balanced diet, moderate exercise, and constant learning of new things can all help us maintain a good memory. In addition, socialization has also been shown to be good cognitive training that promotes regular training of the neurological functions of the brain.

Finally, we should keep a positive attitude. While Parkinson's disease may affect a person's physical and cognitive function, we shouldn't let it become the main focus of our lives. Instead, we should always maintain a positive attitude and keep our days as fulfilling and loving as possible.

In short, Parkinson's disease is related to memory, but we can help maintain a good memory through some measures and methods. In addition, we should also maintain a positive attitude to better deal with various challenges. From this point of view, we need to improve our memory. Cistanche can significantly improve memory, because Cistanche can also regulate the balance of neurotransmitters, such as increasing the level of acetylcholine and growth factors, which are very important for memory and learning. In addition, meat can also improve blood flow and promote oxygen delivery, which can ensure that the brain receives sufficient nutrition and energy, thereby improving the vitality and endurance of the brain.

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Out of the six tested SNPs, we have identified only rs708727 as being associated with an increased risk for PD onset in Slovaks. The minor allele (A) in rs708727 is associated with PD in dominant and completely over-dominant genetic models (ORD = 1.36 (1.05–1.77), p = 0.02, and ORCOD = 1.34 (1.04–1.72), p = 0.02). Furthermore, the genotypic triplet GG(rs708727) + AG(rs823156) + CC(rs61822602) might be clinically relevant despite showing a medium (h ≥ 0.5) size difference (h = 0.522) between the PD and the control populations. 

RandomForest modeling has identified the power of the tested SNPs for discriminating between PD patients and the controls to be essentially zero. The identified association of rs708727 with PD in the Slovak population leads us to hypothesize that this A1 polymorphism, which is involved in the epigenetic regulation of the expression of the ADlinked gene PM20D1, is also involved in the pathobiology of PD (or universally in neurodegeneration) through the same or similar mechanism as in AD.

Keywords:

Parkinson’s disease; Alzheimer’s disease; PARK16; Na+/Mg2+ exchanger; SLC41A1; single nucleotide polymorphism.

1. Introduction

The role of magnesium (Mg) homeostasis (MgH) in the pathophysiology of Parkinson’s disease (PD) is the subject of ongoing research and debate. The broad range of Mg actions in cellular physiology and at the level of the organism substantiates the assumption that disturbed MgH contributes to the degenerative processes associated with PD.

Magnesium is essential for cellular energetics [1,2]. It is required for ATP production, the stabilization of its structure, and its biological activity [1–3]. Overall, it interferes with mitochondrial homeostasis (MH) at various levels, from the structural organization of the mitochondria to various processes of mitochondrial respiration [1–6]. 

The close relationship between MgH and MH is also illustrated by the mitochondria serving as the main reservoir of Mg2+ in the cell [7]. Furthermore, Mg has anti-apoptotic, pro-proliferative, and pro-growth properties, and constituents of Mg homeostatic machinery interfere with cellular Akt/PKB and Erk1/2 pro-survival signaling [8–10].

Neurodegenerative diseases, including PD, are characterized at the cellular level by damage to MH and to the energy stability of the cell resulting primarily from aberrant mitophagy, ER-stress management, retromer function, ubiquitination, and adjacent protein turnover, i.e., processes that are tightly connected under normal physiological conditions [11–13]. 

The role of Mg in these processes is only marginally understood. Nevertheless, both the cytoplasmic pool (secured primarily by the coenzyme TRPM7) and the intramitochondrial/matrix pool (secured by the superconductive Mg2+ ion channel Mrs2) of Mg2+ are known to be particularly important for the maintenance of the membrane potential on the inner mitochondrial membrane (∆ψm) [2,4–6]. 

Any drop of matrix [Mg2+} attributable to the Mg-starvation of the cells, or the dysfunction of Mrs2, induces a depolarization that further triggers mitophagy [14]. Recently, Zhao and colleagues have demonstrated that high glucose induces a drop of intracellular [Mg2+] accompanied by the induction of mitophagy in hFOB1.19 cells (conditionally immortalized fetal osteoblasts, ATCC CRL-11372™) [15].

The cytoplasmic and, indirectly, the intra-organelle (mitochondrial, ER, Golgi) concentrations of Mg2+ are dependent on the Mg2+ transporters, which constitute the Mg2+ transport circuit of the cytoplasmic membrane [6]. These transporters are as follows: (1) the coenzyme TRPM7, the major cellular Mg2+ influx gateway, and (2) the Na+/Mg2+ exchanger (NME) SLC41A1 (further referred as to A1), the major cellular Mg2+ efflux gateway [6,16,17]. 

Indeed, Cornell’s group has hypothesized that the published epidemiological data “support the possibility that mutations in genes relevant to MgH would alter PD risk” but warrant “deeper genetic analyses of PD patients” for confirmation that SLC41A1 (further referred as to A1) and TRPM7 are among these genes [18].

About the possible involvement of NME A1 in the onset and progression of PD, Tucci and colleagues have identified two novel coding variants of genes that are from the PARK16 locus and that are present only in the PD cohort, namely RAB7L1 (c.470A > G; p.K157R) and A1 (c.1049C > T, p.A350V) [19]. The former group of Kolisek has characterized A1 c.1049C > T as a gain of function mutation resulting in “enhanced Mg2+-efflux conducted by A1 variant p.A350V”, which might lead, in the long-term, to “chronic intracellular Mg2+ - deficiency, a condition that is found in various brain regions of PD patients and that exacerbates processes triggering neuronal damage” [20]. 

Lin et al. have subsequently identified a rare loss of function variant of A1 p.R244H in a cohort of 80 patients diagnosed with early-onset PD [21]. The mechanism behind the loss of function of A1 is a matter of speculation, although, Tatarkova and colleagues have recently provided data making it clear that “the presence or absence, and thus the functionality, of A1 influences mitochondrial processes involved in energy production” [1]. Moreover, Li and colleagues have recently associated the A1 variant p.R285Q with PD [22]. 

Further experimental evidence of the possible involvement of A1 in PD has been provided by Lin and coworkers who have shown that Mg sulfate (MgSO4) possibly protects SH-SY5Y cells against the neurotoxicity of 6-hydroxydopamine (6-OHDA) [23]. They have additionally demonstrated that 6-OHDA decreases the expression of A1 (and other magnesiotropic genes) in 6-OHDA-treated SH-SY5Y cells, and that MgSO4 can reverse its decline [23]. The same group has also provided data revealing that, in a rat PD model, 6-OHDA alters the expression of A1/A1 (at both the RNA and protein levels), and that the extent of this alteration is responsive to [MgSO4] [23].

The PARK16 locus comprises five genes, namely SLC45A3, NUCKS1, RAB7L1, A1, and PM20D1 [24]. Its role in the susceptibility to PD has been pointed out by numerous genome-wide association studies (GWAS) and case-control studies. Three A1 single nucleotide polymorphisms (SNP(s)) have been extensively studied concerning their association with PD.

The major G allele of the A1 polymorphism rs11240569 (for characteristics see Table 1) of a Han cohort in China has been shown to reduce the risk of idiopathic PD, with people who have the GG and AG genotypes exhibiting a reduced risk compared with those who have the AA genotype [25]. A similar outcome has been obtained in a study performed with an Iranian cohort [26].

Another A1 SNP, rs708727 (Table 1), has been studied in a UK cohort, but no association between this SNP and PD has been found [19]. However, this SNP has been linked to Alzheimer’s disease (AD) [27].

About PD, rs823156 (Table 1), is probably the most intensely studied but is also the most controversial among the A1 SNPs. This SNP has been associated with PD in cohorts from mainland China [28], Japan [29], and Korea [30], but not in cohorts from Eastern China [31], the north of Spain [32], and Malaysia [33]. Bai and colleagues have predicted, following in silico analyses, that rs823156 as a noncoding variant of A1 “might affect PD risk by altering the transcription factor-binding capability of the genes” [34].

Previously published work has made it obvious that cells regulate the extent of Mg2+ efflux via A1 at the level of proteins and the level of transcription [17,20,35,36]. However, the amount of information about the organization of the promoter of A1 and its transcription-binding capacity is rather scarce [34].

In 2019, we published a study showing that the three aforementioned A1 SNPs are not associated with any susceptibility toward PD in the Slovak population, as demonstrated by the means of frequentist statistics and machine learning [37]. A major limitation of that study might have been the relatively low number of participants in both the PD (150) and the control (120) cohorts. 

Therefore, the aim of this study has been twofold, as follows: (1) to elucidate any possible association of rs11240569, rs708727, and rs823156 in a larger group of PD patients (150 + 358) and control probands (120 + 352), and (2) to sequence the promoter region of A1 in a sub-cohort of PD samples to identify any possible SNPs within the promoter region and to examine their possible association with PD.

2. Results

2.1. Sequencing of SLC41A1 Promoter Region

The Sanger sequencing and sequences analysis was performed in a sub-cohort of 96 PD patients (all from the PD Center in Martin). A fragment of the A1 promoter region was studied, spanning from position 205,814,626 to 205,812,988 on chromosome one. The sequence of the fragment was chosen according to the Genecopoeia database [www.genecopoeia.com/ product/search/view_seq_promoter.php?cid=&type=promoter&prod_id=HPRM53412 (accessed on 2 May 2018)]. 

The gene organization of A1 and its promoter/regulatory sequences is depicted in Figure 1. The sequencing allowed the identification of the following four SNPs in the A1 promoter region: rs9438393, rs56152218, rs61822602, and rs144056491 (Figure 1). Next, we utilized the RFLP strategy to examine rs9438393 (restriction with Hpy166II), rs56152218 (restriction with NIaIII), and rs61822602 (restriction with BmrI) in a sub-group of 100 control samples. The SNP rs144056491 was not examined in the control group because of the lack of a suitable restriction enzyme.

ConSite [38] (Table 2), a web-based tool for finding cis-regulatory elements in genomic sequences, was employed to examine whether the variant (minor) allele for each of the four aforementioned SNPs altered the TF-binding profile of the A1 promoter by rendering a new TF-binding site or by erasing the existing one. As input, we used 33 bp long sequences, one with the reference (major) allele and the other with the variant (minor) allele for each SNP, respectively. 

At rs144056491, which is located within the binding site of transcription factor p50, both the major allele (C) and the minor allele (-, CC, CCC) presumably allow the binding of this transcription factor (Figure 1). The presence of the major allele (A) at rs9438393 might permit the binding of the transcription factor FREAC-4 (Table 2, Figure 1). However, if the minor allele (G) is present, then the FREAC-4 binding site is no longer recognized by the TF-binding predictive software. At the same SNP, the minor allele putatively allows the binding of SP1, which is not the case in the presence of the major allele (Table 2, Figure 1). 

The major allele (T) at rs56152218 putatively allows the binding of Gata2, but according to the prediction, this will not be the case in the presence of the minor allele (C). On the other hand, YY1 might bind the minor C-allelic variant, but not the major T-allelic variant (Table 2, Figure 1). According to the in silico prediction, SNP rs61822602 is not located within any TF-binding sequences (Table 2, Figure 1).

Figure 1. Gene organization of A1 including adjacent upstream 50UTR. According to Ensembl Transcript: SLC41A1-201 ENST00000367137.4, this gene is located on chromosome 1 and consists of 11 exons. Exon 1 represents 50UTR (untranslated region), and exon 2 contains a part of this 50UTR. 3 0UTR is included in exon 11. In our previous study, we studied three SNPs (single nucleotide variants), namely rs11240569, rs708727, and rs823156 in A1 [37]. In this work, we analyzed a sequence (1638 bp in length) located upstream of this gene. 

This sequence covers the 50upstream sequence and, partially, exon 1. According to the UCSC genome browser [39], the sequence is a regulatory region represented by CpG islands (green rectangle). A promoter-like signature (EH38E1415811) and a proximal enhancer-like signature (EH38E14112) (red and orange rectangle, respectively) have been described in this region. 

We have identified four SNPs (rs144056491, rs61822602, rs56152218, and rs9438393) in this sequence. At rs144056491, a search within the reference sequence and then in the sequence with the variant resulted in the identification of a binding site for transcription factor p50. At rs9438393, the search resulted in the identification of a binding site for transcription factor FREAC-4 (the A allele). However, no binding site was detected in the variant sequence (G allele). At the same SNP, the G allele allows the binding of SP1. At rs56152218, the dominant T allele enables the binding of Gata2, and the minor allele that of YY1.

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