The common feature of all proteomic and metabolomic studies regardless the specimen (brain tissue or plasma sample) is alterations observed in energy metabolism, including glycolysis and mitochondria functioning, lipids transformation, and oxidative stress, which integrates changes in various other energy metabolism pathways. In this study, numerous of proteomic and metabolomic elements being involved in pathways touched above have also been found and integrated into the proposed map of schizophrenia and probably they shape the central core of impaired biological processes. The collected proteins, metabolites and putatively associated loci cover 68 biological processes totally, among which only two pathways (hsa04080: Neuroactive ligand-receptor interaction; and hsa04977: Vitamin digestion and absorption) are characterized by intersection of all three layers (Table 3) but include functionally unrelated proteins and metabolites. Some associations are extraordinary relevant to the major hypothesis of the onset and etiology of schizophrenia, including serotonin and dopamine as significantly altered compounds (Table 2), and associated gene polymorphisms (GRM8, PTGS1, GABBR2), covering glutamatergic, serotonergic, GABAergic, and dopaminergic synapse transmission and. Parallel transcriptomic and metabolomic study of post-mortem prefrontal cortex endorses the toxic effect of aberrant glutamatergic transmission on synaptic plasticity and myelinization in patients with schizophrenia26. It should be notices, that the PTGS1 loci was characterized by two distinct SNPs (rs8046 and rs10306166; Table 3) and the resulting protein product is a part of serotonergic and glutamatergic synapse plasticity and transmission (hsa04726; Table 3).
Energy metabolism and lipids metabolism as constants at schizophrenia
The review of proteomes among psychopathology researches indicates only a small fraction of proteins (A2M, APOA1, APOB, APOC1, APOH, C4BPA, etc.) reasonable to schizophrenia27. Evidence exists about relationship with various defensins and interleukins (IL-1ra, IL-8, IL-10, IL-15, etc.), which are differentially expressed at schizophrenia28,29. Most of promising protein markers were detected in this study and joined with the established metabolic compounds (Fig. 3). The most challenging issue is that some of these proteins are also constantly proposed for Parkinson’s and Alzheimer’s diseases that exhibits their weak specificity30,31. Notwithstanding, due to the majority of studies highlights lipids metabolism as generally observed affected pathway at schizophrenia, we assume that its relationship with the cholesterol metabolism (hsa04979) and associated signaling is the most essential reference point due to the interplay between APOM, carrying 9-cis-retinoic acid ligand, and APOD, affecting the RXR/PPAR receptors; both regulate the expression of numerous of apolipoproteins (Fig. 3).
Violation of lipid metabolism and shift in redox balance towards the production of reactive oxygen species (ROS) strongly correlates with APOD regulation32,33. Synthesis and secretion of APOD are inversely dependent on the concentration and duration of exposure to 17β-estradiol34. Therefore, combinatory therapy of antipsychotic drugs with estradiol shows a positive effect on the recovery of patients with schizophrenia due to targeted action on the expression of receptors and transporters of serotonin in the forebrain and due to the decrease of APOD, which is associated with prolactin antagonizing the estrogens production35.
Negative symptoms of schizophrenia are curtained by the complex disturbance of lipids metabolism, oxidative stress, and pro-inflammatory reaction at the prodromal period. We found that the combination of apolipoproteins (APOA2, APOA4, and APOC1) and nonclassical lipocalins (APOE, APOD, APOM, APOH, CLU) tends to moderate declination (Table 1). Several studies reported the predictive power of these apolipoproteins for highly specific differentiation of schizophrenia with specificity above 83%9,36. However, data about the APOE implication are the most ambiguous and contradictive. While many studies review the importance of APOE in organization of glia cytoarchitecture and synapse plasticity, considering APOE polymorphisms as candidates for risk of schizophrenia37, the meta-analysis of 28 studies revealed such association only in Asian population38. In this study, we did not detect APOE as a significantly altered protein, however other members (APOH, APOB, APOC3) of cholesterol metabolism and PPAR signaling were differed compared to the control group (Table 1).
Since we did not find sensitive SNPs of the detected apolipoproteins but the level of these proteins is markedly increased in patients with schizophrenia (Table 1), assumingly other mechanisms implicated in apolipoproteins signal transduction and receptor-mediated binding might be more tightly associated with the disease. The GWAS defined associated SNP (rs61941687) only for SCARB1 gene (Supplementary Appendix E) related to hsa04979 and hsa04975 (Table 3). Despite SCARB1 is a receptor of lipophilic ligands, including phospholipids, there are no credible data about relation of exactly SCARB1 to schizophrenia, albeit changing of phospholipids profile was established in metabolomic assay of patients with schizophrenia and bipolar disorder. However, such changes were found in patients after treatment by lithium and risperidone39,40, which were also used in treatment strategy of our study. Probably, a few detectable in the study phospholipids remained unaltered due to post-processes cleaning of metabolic data from drug-affected and drug-related metabolites (see Supplementary Appendix D).
Governors of oxidative stress and immunoreactivity
Ultimately, dysregulation of energy metabolism and lipids metabolism entails to overproduction of reactive oxygen species and immune response. Dysregulation of redox-responsible proteins, including PRDX6, GFAP, MDH and ATPase subunits, have been repeatedly identified in plasma and prefrontal cortex of patients with schizophrenia41,42,43. Unfortunately, researchers pay little attention to mechanisms instructing the pathogenesis of schizophrenia. We found highly abundant GPX3 that indicates an over-scored ROS generation since the enzyme utilizes end-products of GSP and SOD due to increased lipids peroxidation. Earlier, alterations in GST, A2M, and IL-17 were demonstrated for 25%, 17%, and 20% of patients9. Assumingly this confirms our suggestion about stressful oxidative damage; thus, signs of experienced apoptosis can be expected, among which BCL9 was undoubtedly distinguished (Supplementary Appendix C).
Closer examination revealed progranulin (GRN) as a potential marker of schizophrenia. It regulates inflammation and transport to lysosomes, and acts as an IL-8 inducer. The association between GRN, neurodegenerative diseases (including dementia), and bipolar disorder has been reliably recognized44. At least in murine models, deficiency of GRN provokes autophagy and impaired signaling in neurons45. GRN is a secreted protein and regulated by TFEB46. The protein is delivered and exported to lysosomes after heterodimerization with prosaposin. Some mutations in GRN induce the increased activity of TFEB and upregulation of lysosome-specific proteins together with hyperactivity of the innate immunity47. As a result, neurons and microglia cells include large-size mature lysosomes, which ultimately entails to cell toxicity and rapidly expanding inflammation processes48, both are confirmed by the elevated level of CERU, CLU, and complement factors (Table 1).
Studies showed that GRN deficiency is accompanied by increased expression of IL-6, IL-8 and C4 complement factor33,49. While IL-8 reduces the production of estrogens50, we found estrone among significantly suppressed hormones (Tables 2, 3) involved as in steroidogenesis, as in regulation of dopamine secretion and immune response (Figs. 3, 4). At the same time, C4A, C4B, and C3 are expectedly reduced (Supplementary Appendix C), but the abundance of C4BPA is significantly higher, which may explain the alteration of C4 level. Recent study demonstrated an increased expression of C4 SNP variants and associated escalation of activity of complement system during schizophrenia, which has also been correlated with the reduced synaptic density in post-mortem brains of schizophrenia patients51. Immune system bears a huge number of sex hormones receptors, hence, deficient of estrogens (Table 3) reduces the immune response52.
Circular plot for the Multi Omics connections between the serum-based proteome, metabolome and SNPs recognized in patients with schizophrenia. The external environment of the graph shows the most affected biological pathways where the identified meaningful molecular factors (Table 3) are involved in. The graph size was equal to 554 with assortative value of 0.346. Connections (colored lines, or edges) entering and outputting in/from different pathways (nodes, or vertices) illustrate a track caused by common elements between molecular layers (proteome—red line; metabolome—green line; gene with SNP—blue color) that has been defined in the study (Table 3) and consolidated with the current knowledge about pathophysiological mechanism of schizophrenia. If entity (node) has no connection (edge) with other node, there is no element (protein, metabolite, or gene with SNT), which is shared between two or more distinct pathways, so that element is specifically attributed to certain pathway and makes a loop on itself. The more elements (proteins, metabolites, genes with SNP) are congregated and focused in certain pathways (node), the larger size of centrality can be expected. The number of nodes with the centrality more than 10 (i.e., the number of edges attached to the node) was equal to 30, while the centrality more than 20 refers to 5 hubs. The centrality reflects the level of occurrence of a biological process. In turn, the occurrence has been was estimated through the frequency of identification of the corresponding molecular factors in the studied samples. The larger centrality indicates the higher frequency of the molecular factor identification.
We can assume that the leading role of GRN is limited to delaying the activity of microglia for the implementation of neuroimmune signal transmission if neurons are damaged by oxidative stress. Homozygous GNR murine exposed to MPTP exhibited higher density of damaged neurons53. Besides, the activity of complement system (C3 and C4 variants) contributes to brain endothelial activation and is related to the perivascular inflammation and associated angiogenesis54. Damages, caused by the complement exaggeration, require involvement of microglia to rescue the integrity of the impaired blood–brain barrier and brain microvascular system. Transcriptomic screening of neurons and glia in cerebral cortex displayed the decreased expression of angiogenesis-related genes (ANGPT2, FC = − 1.24, p = 5.80e−5; ANGPTL4, FC = − 1.25, p = 6.39e−6; SPARC, FC = − 1.77, p = 7.55e−9) in patients with schizophrenia55,56. Remarkably, but the most recent GWAS study identified rs592927 loci associated with ANGPTL2 (Supplementary Appendix E) and linked it with the paracrine induction of angiogenesis and acute inflammation reaction57.Therefore this loci was ushered as a promising marker for recognition of the early schizophrenia58.
In a sense, the current scenario intends to violation of lipids metabolism and manifesting of oxidative stress and immune reactivity (Fig. 3). The deficiency of GRN causes the growing secretion of complement factors by microglia and, thus, tightly associates hsa04080 pathway (neuroactive ligand-receptor binding) through all omics-layers (Fig. 4) by collecting PLMN protein, two SNPs (rs2299542 and rs118125840 for GRM8 and GABBR2 glutamate receptors) and metabolites (norepinephrine, dopamine (Supplementary Appendix E), and serotonin (Table 2)). The activity of GRN largely determines the transport to lysosomes, and can designate the increased level of PLMN and GPX3 due to overproduction of ROS. Despite transcriptomic data evidence about the diminished immune response events59, the suppression of microglia by GRN, activation of complement system, and overexpression of GPX3 severely argue for the inflammation in schizophrenic patients.
Is transportation more important than transformation?
Yet, what is the source of pro-oxidants? Our attention focused on the level of FASN as a sign of fatty acid accumulation and the ongoing reduction of triglycerides output, despite previous review noticed the reduced level of polyunsaturated fatty acids but elevated level of lipids peroxidation products60 The source of ROS is emerging from the activity of FASN that requires NADPH and, consequently, increases rate of PPP (Fig. 3). Noteworthy, the highest expression of FASN is observed in brain and liver tissues.
Obviously, the increased activity of FASN can be caused by the impaired fatty acids oxidation and glycolysis. In part, that is confirmed by changing in APOB, which indicates the reduced level of VLDL/LDL and the violation of their transport. But here we faced contradictory uncertainty: SORT is a receptor for both GRN and APOB and participates in the transfer of LDL and its further endocytosis. In conditions of the depleted fatty acids transport, one can expect a decrease of activity and surface density of SORT. Oddly, it has been indicated an increase of SORT in patients with schizophrenia61. Turning to the panoply of evidence-based data and results obtained in our study, we concluded that the root of pathology is underlined in lipids transport rather than in lipids metabolism.
Occasionally, we found SNP (rs11254275) in CUBN loci quite frequently (> 0.88) among studied patients, which permits to locate it together with APOB protein and 7-dehydrocholesterol (vitamin D3) into hsa04977 pathway (Table 3). Due to CUBN is an endocytic receptor, non-synonymous exon polymorphism in CUBN gene might be a reason of alternating lipids and apolipoproteins transport and absorption, thus an increased surface density of SORT is considered as a compensatory mechanism. Furthermore, clinical trials and associated genome studies displayed that some splice-variants of CUBN cause a deficient of vitamin D transport62, whereas C-terminal mutations may induce metabolic syndrome accompanied with severe inflammation63,64. Indeed, the resulting trial has many common features with prediabetic conditions because impaired glycolysis and lipids transport are commonly observed features of patients with diabetes mellitus. Moreover, there are reports showing high risk of IR (insulin resistance) and development of T2DM before long following the first episode of schizophrenia had been registered65,66, albeit we did not recognize difference in proteome and metabolome between resistant patients and those who suffered the first episode of schizophrenia (Supplementary Appendix A). Does it turn out due to above-mentioned reasons that schizophrenia might be a lysosomal disease associated with a malfunction of lipids transport?
Cooperation with steroidogenesis
The molecular harmonization between proteome and metabolome merits attention due to several meaningfully observed compounds (Table 2), which are subdivided into lipids (glycocholic acid (GA), androsterone sulfate (AS), cholesterol sulfate (CS), dehydroepiandrosterone (DHEA)), carbohydrates and their derivatives (sphingosine (C16S), 3-O-sulfogalactosylceramide (3OSG)) and amines (serotonin (SR), thyroxine (T4), normetanephrine (NMN)). A portion of metabolites (CS, AS, GA, DHEA) is epitomized as a sequential transformation of cholesterol (Table 2, Fig. 3) and associated with loci polymorphisms (Supplementary Appendix E) via hsa04975, hsa04979, hsa04976 pathways.
Cooperation of apolipoproteins with FASN, GPX3, and PEDF in steroidogenesis and in the transport of retinoic acid sharply affects the regulation of the HPA axis. By this reason, the alteration of progesterone and pregnenolone (Table 2) assumingly operates as an indicator of psychopathological changes due to their progenitor activity for neurosteroids that stimulate inflammatory and immune responses. Due to AS (androsterone) conjugate is a substrate for DHEA, it shapes a comprehensive relationship between enzymes of the opposite effect (lyase and transferase), and the ability to convert AS into DHEA and further into estrogens (Fig. 3). Deficiency of aromatase impedes the catalytic conversion of androstenedione and testosterone to estradiol, and NADH is compulsory for this reaction and together with NADPH and oxygen is essential in fatty acids oxidation and PPC (Fig. 3). In this respect, we assume that the detected changes of the circulating DHEA and androsterone sulfate conjugate (Table 3) exhibit symptoms severity, persistence to treatment, and cognitive dysfunction in schizophrenia[NO_PRINTED_FORM].
There is evidence that DHEA exhibits dual pro-oxidative and defending properties achieved through the interaction with PPAR receptors67, which are tightly engaged with hsa04024 pathway via cAMP uptake. Admittedly, an increased level of DHEA and consequent stimulation of PPAR/RXR receptors68 leads to increased generation of hydroperoxides. The growing risk of oxidative stress is aggravated by FASN and adiponectin upregulation69, and following triggering of glutathione, GST, G6PDH, and GPX3 all involved in the metabolism of arachidonic acid (hsa00590) and prostaglandins. At the same time, DHEA is a progenitor for androgens, among which AS and testosterone can be transformed into APOD-stimulating regulation of estrogens, which are antagonized by prolactin (Fig. 3).
Evidence suggests the relationship between DHEA, increased GPX3, and decreased proportion of adipose tissue. A larger volume of adipose tissue positively correlates with FASN activity which fosters the DHEA stimulation70. Thus, the accumulation of fatty acids and increased triglycerides, examined in patients with schizophrenia, entails to stimulation of DHEA, AS, their mutual interconversion into estrogens, and subsequent events of lipids uptake via PPAR-α/γ1/γ2 receptors (Fig. 3, Table 2).
Adipocytes represent the main storage of triglycerides as the most oxidized elements. These elements enhance NO generation needed for endothelial permeability and provide protection through the induction of PTGS1. According to GWAS data71, the most significant polymorphisms (rs8046 and rs10306166) were associated with hsa00590 pathway and discovered for cyclooxygenase gene PTGS1 (Table 3, Supplementary Appendix E), necessary for PTGH production as an antagonist of thromboxane due to adenylate cyclase stimulating properties. This, possibly, associate both SNPs with the platelet activation (hsa04611) and lipolysis processes (hsa04923), causing elevated norepinephrine generation (Table 2) in patients with schizophrenia72,73. Malfunctions of PTGH impact the proper action of PTGS1 and fraught with the angiopathy, and the risk of thrombosis largely frequent among patients with schizophrenia. Hence, the selected elements of hsa0059, hsa04611, and hsa04923 pathways and direct relation of PTGS1 to serotonergic synapse plasticity (Table 3) might be considered as ancient factors of schizophrenia etiology.
Harmonization with thyroid hormones
Whether DHEA and AS are further boosted along the path of estradiol synthesis? Indeed, if we assume based on the obtained proteomic data that patients with schizophrenia have a complex sign of oxidative stress, then probably smaller fraction of DHEA and AS intend to aromatization (Fig. 3). Hence, aromatase activity and action on adipose tissue would be reduced, whereas prolactin activity, on the contrary, would be increased, which means stimulation of APOD and amplifies the ROS production. Above and beyond, the increased AS (Table 2) requires SCARB1 as a receptor. Both elements (AS and SCARB1) are members of hsa04976 and hsa04979 pathways, however the rs61941687 polymorphism (Table 3, Supplementary Appendix E) negatively affects the activity of SCARB1 and, this dire consequence is compensated by the increasing density of SORT74.
There is no consensus regarding the role of DHEA and AS in the pathogenesis of schizophrenia. Data are mostly contradictory, depending on either the daily cycle or treatment strategy, or other unpredictable effects75. There is a trend toward a positive association between DHEA and the severity of schizophrenia, which mostly fits the interface highlighted in this study. The PPAR-mediated metabolism of lipids (Figs. 3, 4) is highly dependent on selenium and requires cooperative action of DHEA and thyroid hormones. However, the overproduction of T4 and T3 does not affect the DHEA-dependent PPAR-signaling76. Thereof, T4 deficiency and DHEA overproduction are common symptoms attended by oxidative stress and pro-inflammatory condition at schizophrenia77.
Due to the synthesis of T4 and functioning of GPX3 are determined by selenium, we cannot bypass the potential role of SELENOP, which interconnects GPX3 activity with thyroid hormones releasing78 (Fig. 3, Table 1). Its responsibilities are focused on the transfer of selenium in the tissues, including the brain, and antioxidant functions. The observed elevation (Supplementary Appendix C) can be caused by a higher demand of selenium due to both upregulated GPX3 and T4 biosynthesis on the background of subclinical hypothyroidism.
There have been far few studied about the positive association between thyroid-stimulating hormone (TSH) and prolactin but symptoms of hyperprolactinemia on the background of hypothyroidism are usually observed79. In this study, we found cautioning reduction of L-3,5-diiodotyrosine and thyroxine hormones (Table 2) interconnected with dopamine and catecholamines uptake (hsa00350 and hsa04918 pathways; Fig. 4). But only GPX3 with inclusion of two SNPs (rs8046 and rs10306166) is co-associated with the regulation of thyroid hormones synthesis and considered as an entering point of arachidonic acid metabolism and serotonergic synapse transmission (Table 3). Other studies indicated a relationship between the severity of schizophrenia and the diminished synthesis of T3 andT4, and T4 is lowering more apparent. However, there was no difference in TSH compared to the control group80.
The tacit assumption is that subclinical hypothyroidism is regular for schizophrenia patients, but some research witnesses about its association with antipsychotic medication81. There is no unambiguous opinion, but T4 increases gradually at schizophrenia, although data were related to the first hospitalization and attributed to stressful situation as a consequence of neuroprotective modulation81. Generally, there is a gentle tendency to the decrease of T4, which positively affects the prolactin level in patients with schizophrenia80,81. In turn, prolactin sends a positive effect on APOD and inhibits adiponectin, thus reducing the concentration of aromatic steroids. Due to estradiol inversely correlates with oxidative stress (through FASN and APOD; Fig. 3), it greatly contributes to the increased risk of oxidative stress.
Insofar our concept fits into the deployed proteomic-metabolic framework (Figs. 3, 4), we encouraged further investigation of association between serotonin, tyrosine, T4 and dopamine. It is known that the elevated dopamine entails to the decrease of TSH and T4. This is supported by the data for therapy by the dopamine receptor blockers leading to subclinical hypothyroidism and hyperprolactinemia that causes the elevation of TRH.
There are clinical observations establish a strong negative correlation between 5-hydroxyindolylacetic acid (5-HIAA) with T3 and TSH in patients with schizophrenia. It is essential to understand that T4 and TSH impacts nor serotonin secretion per se but the population (density) of serotonin receptors and, therefore, their vulnerability to serotonin82,83. Substitutional therapy of schizophrenia with TSH and treatment of depression with T4 reliably improves amelioration of patients and delivers a positive effect on the level of serotonin due to the increased perceptivity of 5-HT receptors84. Otherwise, the increase of 5-HIAA is recognized due to a stoichiometric shift between serotonin and its receptors towards the ligand of the receptor.
Normetanephrine as a catecholamine (Supplementary Appendix D), is a deactivated product of norepinephrine, and a product of tyrosine transformation. There are extensive data on the substantial role of catecholamines in the severity of schizophrenia85,86. The axis of norepinephrine and serotonin via diencephalon into the limbic system and the cerebral cortex plays a considerable role emotional component of human’s life. Patients with the compromised noradrenergic transmission are characterized by the increased T3 in brain nuclei since norepinephrine is a T3 co-transmitter in noradrenergic signal transduction87.
Thus, depletion of catecholamines typically accompanies a deficiency of serotonin. Indeed, the level of norepinephrine and normetanephrine is lower in patients with schizophrenia (Supplementary Appendix D). There is considerable evidence that the decrease is caused by the reduced activity of brain monoamine oxidase (MAO-B) in patients with schizophrenia88, which is complementary with our data. In this respect, the role of estrogen in schizophrenia pathogenesis is severely important, since estrogen positively regulates dopamine activity. Thus the deficiency in estrogen obviously entails to the increased catechol-O-methyltransferase activity and, consequently, to the decreased dopaminergic transmission. That is the reason of why, medication in combination with estrogens restores dopamine receptors sensibilization and provides amelioration of psychotic symptoms89.
We admit, that medications used for schizophrenia treatment may occasionally produce a significant impact on the reconstructed image of the molecular event in the proposed connective model. Still, the complete view is doubtful due to the high complexity of schizophrenia pathology; however, the interplay between the functions of lipids transport/metabolism, thyroid hormone synthesis, and steroidogenesis more than merely evident (Fig. 4).