Matrix metalloproteinase 9 gene-MMP9-DNA methylation status in Turkish schizophrenia patients

Introduction

Schizophrenia is a chronic disorder defined by mental ability deficiencies, in perceptions, thought processes, and emotional responses, along with positive and negative symptoms [1, 2]. Although the research on neurobiological features of the disease has been going on for several years our knowledge of the pathological mechanisms of schiozphrenia is still limited. There has been increasing interest in the mechanisms by which established risk factors for psychosis like genetic and environmental influences may affect the brain [1]. Genetic studies have pointed candidate regions in the genome that are thought to play a role in schizophrenia earlier. However, these findings were insufficient to elucidate the etiology of the disease, and this suggested that the schizophrenia risk factors were not caused by the DNA sequence alone [3, 4].

Almost all clinical symptoms of schizophrenia are related to abnormal brain pathways such as disorders in neuronal circuits, synaptic plasticity and glutamatergic neurotransmission in studies focusing on the etiology of the disease [5]. In studies conducted from the past to the present, it has been suggested that genes included in processes like glutamatergic signaling and regulation have an effect on the pathology of schizophrenia [6]. Matrix metalloproteinases (MMPs) in the protease group focus on extracellular degradation of the components of the extracellular matrix, as well as cell adhesion molecules and cytokines and growth factors [7]. The enzymes belong to this family take part in several developmental and disease-related processes [8].

MMPs have emerged in the brain as an influential repository of signaling molecules that regulate the extracellular matrix, which can affect synaptogenesis, synaptic plasticity, and other different processes take part in the central nervous system. Overactivity of MMPs is harmful to tissues. Therefore, expression of the enzymes, maturation of mRNA, distribution, protein release and activation are regulated and often controlled. The best example of MMPs is the MMP9 [7]. The MMP9 gene is a zinc-dependent extracellular endopeptidase and is located in 20q13.12 chromosomal region [2]. Neuronal MMP9 controls the morphology of dendritic spines and plays a role in synaptic plasticity by regulating the function of synapses. Therefore, they are crucial in learning, memory and cortical plasticity. When the enzyme is secreted in inappropriate morphology, it plays a role in different brain disorders like schizophrenia, autism spectrum disorder, neurodegeneration and brain tumors [9].

Recent studies have reported changes in glutamate signaling that lead to abnormal synaptic plasticity in schizophrenia. Accordingly, it has been shown that MMP9 regulates glutamate receptors, and takes part in synaptic plasticity [10].

There are different genetic and epigenetic studies in the literature proving the connection of MMP9 with schizophrenia. However, as far as we know, Gao et al. (2018) conducted the first investigation to provide epigenetic clues for DNA methylation of MMP9 in schizophrenia patients. In this study, differences in DNA methylation patterns between patients with non-deficient schizophrenia and those with incomplete schizophrenia were investigated. They found that DNA methylation at a total of 9 different CpG positions in exon 4 and exon 5 of MMP9 had been significantly lower [11].

Epigenetics is “the study of heritable changes in gene expression that occur without changes in DNA sequence” [12]. Epigenetic modifications provide a mechanism that can alter genome function and allow gene activity states to propagate from one generation to the next [13]. In addition to genetic studies, increasing studies in the epigenetics field show that epigenetic mechanisms regulate gene expression by methylation of DNA, modifications on histones and also non-coding RNAs have great impact on the etiology of schizophrenia [14].

Studies conducted in this direction have reported DNA methylation changes in the candidate genes namely reelin (RELN), catechol-O-Methyltransferase (COMT), early growth response 1 (EGR1), gamma-aminobutyric acid type A receptor subunit beta2 (GABRB2), and dopamine transporter gene (DAT1) in postmortem brains or peripheral blood samples of patients [15], [16], [17], [18], [19]. Epigenetic mechanisms are gaining increasing acceptance with increasing evidence that they have part in the etiology and pathophysiology of schizophrenia. Therefore, epigenetic studies are gaining importance in schizophrenia [20]. Whether the MMP9 DNA methylation pattern is associated with epigenetic changes in schizophrenia is unclear and further studies are needed.

DNA methylation is an important factor influencing gene expression, and it is unclear whether MMP9 methylation is associated with epigenetic modifications that potentially affect the pathogenesis of schizophrenia. Increased MMP9 level is associated with abnormally long and thin dendritic spines and impaired synaptic plasticity abnormalities and MMP9 has been implicated repeatedly in the pathophysiology of schizophrenia [2]. For this purpose, in our study, we aimed to compare methylation status of MMP9 in patients and healthy controls. To analyze the methylation differences we performed the pyrosequencing method to on the seventh exon of MMP9, which is rich in CpG regions and to our knowledge which wasn’t analyzed in any other population before. Our hypothesis was that there would be a change in DNA methylation patterns of MMP9 in schizophrenia patients comparing to healthy individuals.

Materials and methods

Results

Discussion

In our study, it was shown that the epigenetic pattern of MMP9 of peripheral blood cells was different between schizophrenia and healthy control groups. To best of our knowledge, there is only one study in the literature reporting MMP9 gene exon 4 and 5 DNA methylation differences in schizophrenia patients [11], and only Chinese population was included. There is no study in the literature reporting the differences in DNA methylation of MMP9 exon 7 in schizophrenia patients in the Turkish population or in any other populations. Therefore, our study is the first to report DNA methylation of this region of MMP9 gene in schizophrenia patients.

We hypothesized that exon 7, containing 5 different CpG positions, would represent a potential target for DNA methylation of potential regulatory region of MMP9, and that the change in MMP9 methylation level may contribute to the disease etiology and pathophysiology. We compared the methylation status of 5 CpG regions of MMP9 exonic region in peripheral blood cells of schizophrenia patients and healthy individuals. There were significant differences in the mean methylation of exon 7 and three different positions in the DNA methylation pattern of MMP9 between 40 schizophrenia patients and 32 controls. However, as a result of the analysis of clinical features with methylation value, no significant difference was found. Eventhough we couldn’t find a significant correlation between the methylation status of the gene and the demographic data like age, gender, living environment, age of onset and etc., this may not be the real indicator of the chemical events happening in the adult brain or during brain development.

Both mRNA and protein expression of MMP9 is at low levels in different parts of the brain, predominantly neurons, cerebral cortex hippocampus, and cerebellum. The editing of the MMP9 takes place through multi-step and complex processes. It is vital to regulate enzyme activity very precisely, as proteolytic activity will be harmful to the tissue [9]. Dysfunction of dendritic spines, -a biological feature unique to schizophrenia-plays an important role in the pathology of the disease. MMP9 has been shown to mediate morphological changes in dendritic spines after synaptic stimulation and may potentially play a role in dendritic spine pathology in schizophrenia [10].

There are studies in the literature proving the connection of MMP9 with schizophrenia. Studies showed that MMP9’s C1562T gene promoter polymorphism is associated with schizophrenia [21, 22]. One piece of evidence regarding the contribution of MMP9 to the pathogenesis of schizophrenia is the study of Domenici et al. who reported that it is among the most elevated proteins in patients with schizophrenia. One study showed that MMP9 mRNA levels in peripheral blood samples were strongly regulated by drug therapy in patients [23]. In a study examining MMP9 levels in 2021, patients were found to be higher than the control group, and it was reported that MMP9 increased the risk of cognitive impairment in patients with schizophrenia [22, 24]. In addition to polymorphism studies, plasma levels of MMP9 and its endogenous inhibitor TIMP metallopeptidase inhibitor 1 (TIMP-1) were investigated and found to be increased in schizophrenia patients [25]. Brain derived neurotrophic factor (BDNF) and proBDNF, which contribute to the pathophysiology of schizophrenia, can be converted to their mature form by MMP9. In a study conducted in this direction, a significant relationship was found between BDNF and MMP9 levels in patients with schizophrenia, but no difference was found between healthy controls [26]. It has been reported that MMP9 mRNA levels in peripheral blood mononuclear cells are strongly regulated by drug treatment in patients with schizophrenia [23]. Since epigenetic mechanism namely DNA methylation has huge effect on gene expression and given fact that gene expression levels of MMP9 is changing in patients the methylation status of the gene will directly related with that issue.

We also analyzed the relationship between methylation patterns and the antipsychotic therapy and in line with the literature our results showed that the negative symptoms improved with the antipsychotic treatment however when we checked the correlation between methylation pattern we failed to find an association. But still this association should be investigated deeply related with the chemical effects of drugs on epigenetic changings in future studies.

In addition to genetic studies in the field of schizophrenia, this study took DNA methylation pattern studies in schizophrenia one step further. However, the available findings are not conclusive and there are limitations that should be noted. First, we did not examine gene expression or protein levels of MMP9, so it is difficult to conclude whether DNA methylation changes related to MMP9 match major changes in cellular expression of neural connectivity. Secondly, we used peripheral blood cells in our study since they are easier to reach, but since schizophrenia is a disorder that affects brain pathology and biochemistry, working with post-mortem brain tissues will give more accurate results. Finally, in this study, we were only able to examine the CpG regions located in exon 7, but the study which includes other exons of MMP9 will give a better idea about the DNA methylation pattern of MMP9.

This study is a study aimed to invesitgate the epigenetic contibutions of MMP9 to the pathology of schizophrenia. All studies in the literature on DNA methylation patterns in schizophrenia asks the question “Is DNA methylation a cause or a consequence of schizophrenia?” which still has not been answered. It does not seem possible to answer this question in the short term. Identification of peripheral blood and post-mortem epigenetic biomarkers, including patient families, is necessary to answer this question. By investigating more on DNA methylation patterns in patients who are on drug therapies we will gain more insights to our understanding about the contribution of epigenetic factors to schizophrenia. So this study is the first step to this concept.