Hydrogen sulfide and polysulfide levels in cerebrospinal fluid and plasma of patients with schizophrenia, Alzheimer’s disease, Parkinson’s disease, and in relation to cigarette smoking

Hydrogen sulfide and polysulfide levels in cerebrospinal fluid and plasma of patients with schizophrenia, Alzheimer’s disease, Parkinson’s disease, and in relation to cigarette smoking | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hydrogen sulfide and polysulfide levels in cerebrospinal fluid and plasma of patients with schizophrenia, Alzheimer’s disease, Parkinson’s disease, and in relation to cigarette smoking Hideo Kimura, Yuka Kimura, Kotaro Hattori, Yoshie Omachi, Tadashi Tsukamoto, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7804547/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hydrogen sulfide (H 2 S) and polysulfides produced by enzymes regulate neuronal transmission and protect neurons against oxidative stress. Abnormalities in their levels have been implicated in the pathophysiology of schizophrenia (SZ), Alzheimer’s disease (AD), and Parkinson’s disease (PD). However, the levels of these molecules in the cerebrospinal fluid (CSF) and plasma (PLA) obtained from the same individuals with or without smoking habits have yet to be comprehensively studied. Here, we showed that the levels of neuroprotective H 2 S and polysulfides in the CSF were significantly decreased in patients with AD and SZ compared with those in control individuals, suggesting that neuronal activity is not well regulated and that neurons are inadequately protected in both diseases. In contrast, in PD, the levels of these molecules increase specifically in the PLA to unfavorable levels, suggesting peripheral abnormalities, including inflammation. The increased levels of these molecules in PD were restored by smoking habits to the levels in control non-smokers, suggesting that smoking may be linked to a lower risk of developing PD. H 2 S and polysulfides play important roles in the pathophysiology of psychiatric and neurodegenerative diseases, and therefore, are potential therapeutic targets. Biological sciences/Neuroscience/Diseases of the nervous system/Schizophrenia Biological sciences/Neuroscience/Diseases of the nervous system/Parkinson's disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Indroduction Hydrogen sulfide (H 2 S) is produced by enzymes, including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST), which also produce polysulfides H 2 S n (n ≥ 2) and cysteine persulfide (CysSSH) 1 – 3 . Several other enzymes can also produce polysulfides, including sulfur quinone oxidoreductase (SQR), superoxide dismutase (SOD), myoglobin, catalase, myeloperoxidase, and cysteinyl tRNA synthetase (CARS) 3 . H 2 S is metabolized in the mitochondria by SQR and sulfur dioxygenase to sulfite, which is further metabolized by rhodanese to thiosulfate 4 . H 2 S and polysulfides exert various physiological effects 1 . In the nervous system, H 2 S and polysulfides facilitate the induction of hippocampal long-term potentiation, a synaptic model of memory formation, by enhancing the activity of N-methyl-D-aspartate (NMDA) receptors, facilitating the release of neurotransmitters such as glutamate, GABA, D-serine, a co-agonist of NMDA receptors, and activating transient receptor potential ankyrin 1 (TRPA1) channels in astrocytes, which regulate synaptic activity through the release of gliotransmitters 5 – 7 . H 2 S and polysulfides also protect neurons from oxidative stress called oxytosis, which has recently been proposed to be closely related to ferroptosis 8 , 9 . Disruptions in H 2 S/polysulfide production, as well as in the function of the cystine/glutamate antiporter (Xc − ), a key target in oxytosis and ferroptosis, have been implicated in the pathogenesis of Alzheimer’s disease (AD), Parkinson’s disease (PD), and other neurological disorders, including schizophrenia (SZ) 10 , 11 . Polysulfides release Nrf2 from the Keap1/Nrf2 complex to upregulate antioxidant genes and protect cells from oxidative stress 12 . Low concentrations of H 2 S exhibit beneficial effects, whereas high concentrations are toxic. H 2 S levels are increased by its overproduction or deficiency of its metabolizing enzymes. For example, CBS, encoded on chromosome 21, which is present in triplicate in individuals with Down’s syndrome, is overexpressed, leading to excessive H 2 S production that inhibits mitochondrial electron transport, oxygen consumption, and ATP production 13 . Another example is sulfur dioxygenase, an H 2 S metabolizing enzyme, which is deficient in ethylmalonic encephalopathy, leading to excessive H 2 S levels that suppress cytochrome c oxidase in the muscle and brain 14 . SZ is a chronic and severe mental disorder that affects thoughts, feelings, and behaviors. The symptoms fall into three categories: positive, negative, and cognitive. Both excess and deficiency of H 2 S and polysulfides have been proposed to be involved in the pathogenesis of SZ. H 2 S levels in the PLA were significantly lower in patients with SZ than in normal individuals, and a positive association was observed between H 2 S levels in the PLA and working and visual memory 15 . Thiol homeostasis was disrupted in such patients due to a shift from sulfide (reduced) to disulfide bond formation (oxidized) 16 In contrast, 3MST levels in SZ were positively correlated with symptom severity scores 17 . AD is a chronic neurodegenerative disease that affects cognitive function and memory. The cause of the disease is mostly sporadic, with fewer familial diseases. H 2 S suppressed the activities of β-secretase and γ-secretase to prevent the production of amyloid β-protein via activation of the PI3K/Akt pathway in amyloid precursor/presenilin 1 transgenic mice 18 . H 2 S suppressed tau hyperphosphorylation, which generated neurofibrillary tangles by inhibiting glycogen synthase kinase 3β (GSK3β) 19 . CSE levels were decreased in postmortem human brains and AD mouse models with TauP301L, an amyloid precursor protein with a Swedish mutation, and presenilin 1M146V 19 . Another AD mouse model with five familial AD mutations, in which there was no significant difference in the expression levels of CBS, CSE, CARS1, and CARS2, showed lower levels of total polysulfides and protein polysulfides in the brain than in the wild-type mouse brain; however, no significant differences in the levels of GSH, GSSH, and H 2 S were observed 20 . PD is a neurodegenerative disorder that causes motor dysfunctions. Parkin and α-synuclein are associated with pathophysiology of PD. Parkin is an E3 ubiquitin ligase that ubiquitinates diverse substrates and is responsible for the clearance of misfolded proteins, including α-synuclein. Parkin activity was suppressed by S-nitrosylation, thereby decreasing its protective effect in the brains of patients with PD 21 . In contrast, parkin existed in active and persulfidated (S-sulfhydrated) state in the brains of healthy individuals 22 . H 2 S attenuated neuronal apoptosis in the substantia nigra in a rat model of PD 23 . The intestinal microbiota is involved in neuroinflammation in PD, and H 2 S producing gut bacterium Desulfovibrio is significantly increased in PD compared to that in normal individuals and is positively correlated with disease severity 24 , 25 . Cigarette smoke contains H 2 S, polysulfides, and related sulfur-containing molecules 26 . Smoke produced by one cigarette contains 31.6 µg (0.93 µmol) H 2 S, 40.7 µg (0.68 µmol) carbonyl sulfide, which converts to H 2 S and CO 2 in the presence of water, and 17.6 µg (0.19 µmol) methyl disulfide. Considering that the endogenous concentrations in the brain are in the range of 14 nM–3 µM for H 2 S and 0.2 µM for H 2 S 2 , the acute effect of cigarette smoke is indispensable 6 , 27 . Although H 2 S and polysulfides inhaled from cigarette smoke may be immediately metabolized and may not remain in the body, smoking habits exert various effects on SZ, AD, and PD by altering the endogenous production and metabolism of these molecules. SZ is frequently comorbid with smoking behaviors. Case-control and cohort studies have shown that smoking behaviors cause significantly higher risk of SZ compared with that in non-smokers 28 . Meta-analyses have also shown that smokers have a relatively higher risk of developing SZ than that of non-smokers 29 , 30 . Even a causal role of smoking in the development of SZ has been proposed 31 . An increased prevalence of cigarette smoking is characteristic of individuals with SZ 32 . The microbiome, including bacteria and bacteriophages, influences SZ through neurological and immunological mechanisms. Bacteriophage levels are also influenced by cigarette smoking 33 . Epidemiological evidence from meta-analyses and cohort studies indicates that smoking is associated with a significantly increased risk of AD pathology 34 – 36 , while some prospective cohort studies and Mendelian randomization analyses have shown no association between smoking and AD neuropathology 37 , 38 . Smoking potentially affects the diversity, composition, and abundance of the microbiota and increases anaerobic bacteria and pathogens, leading to the onset and progression of AD 39 . In contrast to AD and SZ, smoking has been shown to be associated with a decreased risk of PD 40, 41 . Although PD smokers are more likely to die from smoking-related cancers than PD non-smokers, they tend to have fewer deaths from neurological causes 42 . Nicotine is the dominant candidate underlying the reduced risk of PD; however, randomized controlled clinical trials of nicotine in PD have failed to demonstrate its benefit on motor function 43 , 44 . Recent findings suggest that non-nicotine components in cigarette smoke may contribute to this protective effect 45 , 46 . Cigarette smoke can alter the composition of microbiota to mitigate intestinal inflammation, leading to reduced misfolding of a-synclein in the enteric nervous system 47 . The blood-brain barrier (BBB) separates neuronal environment from peripheral blood. The molecular size threshold for passive diffusion across the BBB is 400–600 Da, and lipophilicity determines the effectiveness of passive diffusion across the BBB 48 – 50 . An early pathological hallmark of AD is BBB dysfunction, which is characterized by barrier leakage 51 . Accumulating evidence has revealed BBB dysfunction in patients with PD and SZ 52, 53 . The present study examined the levels of H 2 S, H 2 S 2 , H 2 S 3 , CysSSH, cysteine (CysSH), GSH, GSH persulfide (GSSH), GSH trisulfide (GSSSH), thiosulfate, and sulfite in both CSF and PLA obtained from the same individuals. Even the largest molecule examined in this study, GSH (MW: 307) and its persulfidated counterparts, can pass through the BBB, but the CSF to PLA ratio of some of these molecules differed significantly in individuals with SZ, AD, and PD compared with healthy controls. The effects of smoking behaviors differed significantly among these diseases. Results To examine the pathophysiological roles of H 2 S, polysulfides, and related molecules in SZ, AD, and PD, as well as the effects of smoking habits in these neurological diseases, the levels of H 2 S, H 2 S 2 , H 2 S 3 , CysSH, CysSSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in the CSF and PLA obtained from the same individuals were examined. The baseline characteristics of the study participants and the type of diseases are shown in Table 1, and the age distribution of the participants is shown in Fig. 1. Individuals 35–56 years old were grouped as young individuals and 57–81 years old as old individuals. Since it has been reported that aged cells contain fewer persulfidated molecules due to the loss of H 2 S producing enzymes, including CSE and 3MST 54 , the levels of H 2 S, polysulfides, and related molecules in the CSF and PLA of control young non-smokers were compared to those of their older counterparts. The levels of H 2 S, H 2 S 2 , H 2 S 3 , CysSSH, CysSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in both CSF or PLA were not significantly different between control young and old non-smokers (Fig. 2 and Supplementary Fig. S2). The effects of smoking habits on the levels of these molecules in control young and old smokers were examined by comparing them with those in corresponding control non-smokers. CysSSH levels in the CSF of control old smokers were approximately 0.5 fold of those of control old non-smokers and approximately 0.6 fold of those of control young smokers (Fig. 2b, g), while GSH and CysSH levels were approximately 2.2- and 1.7-fold higher in control old smokers than in control young smokers (Fig. 2e-g). In contrast, the levels of H 2 S, H 2 S 2 , and CysSSH in the PLA of control old smokers were approximately 4.4-, 8.2-, and 5.5-fold, respectively, higher than those in the corresponding non-smokers, and H 2 S 2 and CysSSH levels were approximately 3.3- and 2.1-fold higher than those in control young smokers (Fig. 2a-c, g). These observations suggest that smoking habits have a greater influence on old individuals rather than on young individuals. Considering these observations and the age distribution of donors, data obtained from patients with SZ were compared with those of control young individuals, whereas those from patients with AD and PD were compared with those of control old individuals. Data from two patients with SZ older than 57 years (age of 60 and 65 years) and one patient with PD younger than 56 years (age of 52 years) were not included in the present analysis because the ages of these individuals deviated from the defined range (Fig. 1). H 2 S and polysulfides in SZ The levels of H 2 S, polysulfides, and related molecules in the CSF and PLA of patients with SZ were compared with those of control young individuals. The levels of H 2 S (~0.6 fold), H 2 S 2 (~0.4 fold), H 2 S 3 (~0.5 fold), and CysSSH (~0.6 fold) were lower in the CSF of SZ non-smokers than in those of control young non-smokers, while there were no significant differences in the PLA levels (Fig. 3a-d). No significant differences in the levels of CysSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in both CSF and PLA of SZ non-smokers were observed compared with those of control young non-smokers (Fig. 3e, f, and Supplementary Fig. S3). Since the CSF is the extracellular environment of neurons and glia in the brain, these observations suggest that decreased levels of H 2 S, H 2 S 2 , H 2 S 3 , and CysSSH in the CSF are associated with the pathophysiology of SZ. The effects of smoking habits on the levels of H 2 S, polysulfides, and related molecules in SZ were examined. Although CysSSH levels in the CSF of SZ non-smokers were significantly lower (~0.6 fold) than those of control young non-smokers as described previously (Fig. 3b), those of SZ smokers were almost similar to those of control young non-smokers (~0.9 fold) (Fig. 3b). H 2 S levels in the CSF of SZ smokers showed a similar result (from ~0.6 to ~0.7) (Fig. 3a). The recovery of H 2 S and CysSSH levels in the CSF by smoking habits to almost those of control individuals may be related to the smoking preference of patients with SZ (Fig. 3g). In contrast to the CSF, the levels of H 2 S (~3.5 fold), H 2 S 2 (~2.8), and CysSSH (~3.4) in the PLA of SZ smokers were significantly higher than those of SZ non-smokers and control young non-smokers (Fig. 3a-c). These observations indicate that the levels of H 2 S and polysulfides are differently regulated in the CSF and PLA, and that smoking habits influence this regulation. H 2 S and polysulfides in AD Changes in the levels of H 2 S, polysulfides, and related molecules in the CSF and PLA of patients with AD were compared with those of control old individuals. The levels of H 2 S (~0.7 fold) and CysSSH (~0.6) in the CSF of AD non-smokers were lower than those of control old non-smokers, while no significant differences were observed in the PLA levels (Fig. 4a, b). These observations suggest that decrease in both molecules in the CSF may be related to the pathophysiology of AD, similar to that of SZ. There were no significant differences in the levels of CysSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in both CSF and PLA of patients with AD compared with control old individuals (Fig. 4e, f, Supplementary Fig. S4). Smoking habits had no significant effect on the levels of H 2 S, H 2 S 2 , and CysSSH in the CSF of patients with AD, while these levels were approximately 2.2-, 2.6-, and 2.1-fold, respectively, higher in the PLA of AD smokers than those in AD non-smokers and control old non-smokers (Fig. 4a-c). Although the effect of smoking habits on the CSF of patients with AD was different from that of patients with SZ, the effect on the PLA was similar between AD and SZ (Fig. 3g and 4g). H 2 S and polysulfides in PD The levels of H 2 S, polysulfides, and related molecules were measured in the CSF and PLA of patients with PD and compared with those of control old individuals. No significant differences in the levels of H 2 S, H 2 S 2 , and CysSSH were observed in the CSF, while the PLA levels of these molecules were significantly higher in PD non-smokers than in control old individuals (approximately 3.4-, 4.8-, and 3.1-fold, respectively) (Fig. 4a-c). These observations suggest that peripheral inflammation and intestinal microbiota may be involved in the pathophysiology of PD. Changes in these molecules in the CSF and PLA of patients with PD were strikingly different from those in SZ and AD (Fig. 3g and 4g). The association between smoking habits and a reduced risk of PD is among the most robust environmental and lifestyle correlations observed in neuroepidemiology 45 . Smoking habits significantly increased the levels of H 2 S 3 in the CSF (~1.5 fold) compared with PD non-smokers, control old smokers, and control non-smokers (Fig. 4d). In contrast, in the PLA, the levels of H 2 S, H 2 S 2 , and CysSSH in PD non-smokers (approximately 3.4-, 4.8-, and 3.1-fold, respectively) were higher than those in control old non-smokers, and these were decreased by smoking habits to approximately 1.5-, 1.1-, and 1.4-fold, respectively, of control old non-smokers (Fig. 4a-c, g), suggesting an association between smoking habits and a reduced risk of PD. The effects of smoking on these molecules in PD were significantly different from those observed in SZ and AD (Fig. 3g and 4g). H 2 S and polysulfides distribution between CSF and PLA The BBB separates the neuronal environment from the peripheral blood and plays a crucial role in maintaining homeostasis of the central nervous system. BBB dysfunction has been reported in patients with SZ, AD, and PD 51-53 . To understand the specificity of the BBB to H 2 S, polysulfides, and related molecules in these diseases, the distribution of these molecules between the CSF and PLA obtained from the same individuals was examined. The levels of H 2 S, CysSH, and CysSSH (as shown by the slopes of the lines) were higher in the PLA than in the CSF (Fig. 5a-d, i, j), whereas those of GSH and sulfite were lower in the PLA (Fig. 5k, l, o, p). H 2 S 2 , H 2 S 3 , thiosulfate, GSSH, and GSSSH were almost equally distributed between the CSF and PLA (Fig. 5e-h, m, n, and Supplementary Fig. S5). The distribution of H 2 S and CysSSH between the PLA and CSF were not significantly different in SZ non-smokers compared with control Young non-smokers (Fig. 5a, c, and Supplementary Fig. S7a, b). H 2 S and CysSSH were more inclined to PLA (approximately 1.5- and 2.1-fold, respectively) in AD non-smokers than in control old non-smokers (Fig. 5a, c, and Supplementary Fig. S8a, b). In contrast, the localization of H 2 S and CysSSH was significantly more inclined to PLA than to CSF (approximately 3.4- and 4.2-fold, respectively) in PD non-smokers than in control old non-smokers (Fig. 5a, c, and Supplementary Fig. S8a, b). Smoking habits affect older individuals more than their younger counterparts. The PLA/CSF ratios of H 2 S and CysSSH in control young smokers were approximately 2.3- and 2.0-fold of those in control young non-smokers, while those in control old smokers were approximately 5.7- and 14.0-fold of those in control old non-smokers (Fig. 5a-d, and Supplementary Fig. S6a, b). Smoking habits induced shifts in the localization of H 2 S and CysSSH from the CSF to the PLA in SZ (PLA/CSF: approximately 3- and 2.5-fold, respectively) (Supplementary Fig. S7a, b). In contrast, the localization of both molecules shifted less to PLA due to smoking habits in PD (PLA/CSF: approximately 0.4-fold each), which almost recovered to the PLA/CSF ratio of control old non-smokers (Fig. 5a-d and Supplementary Fig. S8a, b). These observations suggest that not only the levels of H 2 S and polysulfides but also their localization between the CSF and PLA have specific inclinations in SZ, AD, and PD. The difference in distribution may be caused by changes in BBB function and peripheral inflammation, as well as a result of smoking habits. Discussion The present study showed that the levels of H 2 S, H 2 S 2 , and CysSSH are distinctively altered in SZ, AD, and PD and are affected by smoking habits. In SZ and AD, the levels of H 2 S and CysSSH significantly decreased in the CSF but not in the PLA, compared with those in the control group (Figs. 3, 4). In contrast, in PD, the levels of these molecules were significantly increased only in the PLA. The effects of smoking habits were divided into two groups: SZ and AD vs. PD. Smoking habits changed the endogenous levels of these molecules and their distribution between the CSF and PLA, suggesting a potential change in BBB function and involvement of peripheral inflammation. The levels of H 2 S, H 2 S 2 , H 2 S 3 , and CysSSH in the CSF of individuals with SZ were significantly lower than those of control individuals (Fig. 3a-d). As the CSF is the extracellular environment of neurons and glia in the brain, changes in the levels of H 2 S and polysulfides in the CSF may directly affect the activity of neurons, and therefore, associated with the pathophysiology of diseases of the central nervous system. Modulation of the cystine/glutamate antiporter Xc - expression has been associated with many neurological and psychiatric disorders, including SZ, AD, and PD 55, 56 . Xc - imports cystine, which is reduced to CysSH in cells, while exporting glutamate from the cell. The hypoglutamatergic hypothesis concerns suppression of the activity of Xc - as a contributory factor to the development of SZ 57 . H 2 S and polysulfides enhance the activity of Xc - 8, 58, 59 . These molecules also regulate the release of neurotransmitters such as GABA, glutamate, and D-serine. Abnormalities in the balance between glutamatergic and GABAergic neuronal activities have been implicated in the pathophysiology of SZ 6, 60 . 3MST-knockout rats, which have lower levels of H 2 S, H 2 S 2 , H 2 S 3 , and CysSSH in the brain, exhibit SZ-like hyperlocomotion following the administration of MK-801, a non-specific NMDA receptor antagonist, compared with wild-type rats 6 . These observations suggest that decreased levels of H 2 S and polysulfides in the CSF are likely involved in the pathophysiology of SZ. The levels of H 2 S and CysSSH in patients with AD were significantly lower in the CSF than in those of control individuals, whereas no significant differences were observed in the levels of these molecules in the PLA, similar to SZ (Fig. 4a, b, g). H 2 S and polysulfides protect neurons from oxidative stress and regulate neuronal transmission 6, 8, 58, 59 . The levels of CSE and persulfidation, which add sulfur to the thiol of cysteine residues, were decreased in postmortem brains of patients with AD. H 2 S produced by CSE prevented pathological phosphorylation of tau by persulfidating GSK3b and inhibited its catalytic activity in an AD mouse model 19 . These observations suggest that a lack of H 2 S and CysSSH in the CSF is involved in the pathophysiology of AD. In PD, the levels of H 2 S, H 2 S 2 , and CysSSH in the PLA were significantly higher than those in control individuals (approximately 3–5 fold), while no significant difference were observed in the CSF levels (Fig. 4a-c, g). This is a critical difference in the distribution of H 2 S and CysSSH in PD compared with that in SZ and AD, where the levels of these molecules decreased in the CSF but did not change in the PLA (Fig. 3 and 4). PD pathophysiology has an influence on regions outside the brain, including peripheral blood 61 . Circulatory system may contribute to PD pathogenesis through the spread of a-synuclein, and BBB allows a-synuclein to infiltrate in the brain 62 . More than 99% of a-synuclein is present in erythrocytes in peripheral blood, and erythrocytes express 3MST, which can produce H 2 S and polysulfides 63-65 . H 2 S/polysulfides persulfidate and activate parkin, which is also expressed in peripheral blood lymphocytes, thereby promoting the clearance of misfolded proteins, including a-synuclein 22 . The intestinal microbiota is involved in neuroinflammation in PD 25 . Numerous pro-inflammatory cytokines, such as IL-6, TNF, and IL-2, are increased in the blood of patients with PD 66 , and PD blood monocytes have a higher proliferative capacity than those of control individuals 67 . The levels of H 2 S and polysulfides, which can mitigate inflammatory stress and enhance the activity of Xc- interacting with blood in the BBB to protect neurons at physiological concentrations, are increased to unfavorable levels in the PLA of patients with PD 8, 68, 69 . The effects of smoking habits were considerably non-consistent between patients with SZ and AD, and those with PD. SZ is reportedly associated with smoking behaviors. Meta-analyses have shown evidence of an increased relative risk of SZ in smokers compared with that in non-smokers 70 . The present study shows that the levels of H 2 S, H 2 S 2 , and CysSSH in the CSF decreased in SZ and were increased with smoking habits to levels that were not significantly different from those of control non-smokers (Fig. 3a-c, g), suggesting that it may be related to the preference of patients with SZ to smoking. In SZ non-smokers, the levels of H 2 S, H 2 S 2 , and CysSSH in the PLA, which were not significantly different from those of control individuals, increased with smoking habits to levels more than two times those of control non-smokers (Fig. 3a-c, g). Chronic peripheral inflammation is associated with the pathophysiology of SZ and has been reported to be associated with smoking preference 71 . The levels of H 2 S and polysulfides increased to suppress peripheral inflammation but reached unfavorable levels. Meta-analyses and cohort-based studies have indicated that smoking is associated with a significantly increased risk of AD neuropathology and associated dementia 36, 72, 73 . The present study showed that the levels of H 2 S, H 2 S 2 , and CysSSH in the CSF were not significantly altered by smoking habits, while those in the PLA were increased by more than two times those of control non-smoking individuals, similar to SZ (Fig. 4a-c, g, and 3g). Smoking potentially affects the diversity, composition, and abundance of microbiota, leading to the onset and progression of AD 39 . Smoking also activates immune cells and induces vascular inflammation, including leukocyte infiltration, in AD 74, 75 . Smoking habits have been associated with vascular endothelial dysfunction and chronic peripheral inflammation in AD and SZ by increasing IL-1b and TNFa 71, 76, 77 . Although H 2 S and polysulfides suppress inflammatory responses and exert cytoprotective effects against oxidative stress by activating Xc - and Nrf2 8, 12 , the levels of these molecules increase to unfavorable levels in AD, similar to SZ. In contrast to SZ and AD, smoking has been reported to be associated with a reduced risk of developing PD 46 . As randomized controlled clinical trials of nicotine in patients with PD have failed to demonstrate a benefit for motor function 43 , non-nicotine components in cigarette smoke have been suggested to contribute to this protective effect 46 . H 2 S 3 levels were significantly increased (~1.5 fold) in the CSF of smokers with PD (Fig. 4d, g). Polysulfides activate the Nrf2 pathway and Xc - to protect cells from oxidative stress 8, 12 . High concentrations of polysulfides and H 2 S inhibit the activity of monoamine oxidase, which degrades dopamine, to effectively preserve dopamine in the brain 78, 79 . In contrast to SZ and AD, smoking habits decreased the levels of H 2 S, H 2 S 2 , and CysSSH in the PLA of patients with PD to less than half of those in PD non-smokers and almost equal to the levels in control non-smokers (Fig. 4a-c, g). Cigarette smoke can modify the composition of microbiota to mitigate intestinal inflammation, thereby reducing misfolding of a-synclein in the enteric nervous system 47 . Smoking habits also alter the physicochemical properties of erythrocyte membranes, which may decrease the levels of a-synuclein in the blood and overexpression of H 2 S and polysulfides in PD 64, 80 . These effects of smoking may be related to its observed association with a reduced risk of PD. Emerging evidence indicates that BBB dysfunction is a key pathophysiological factor associated with tight junction abnormalities and endothelial dysfunction in SZ 53 . Recent clinical data have emphasized the association between BBB leakage and cognitive decline in patients with AD 51, 81 , and BBB alterations in PD 82 . The most commonly used clinical parameter to characterize barrier leakage is the CSF-to-serum ratio. In healthy individuals, the levels of blood-borne proteins, such as IgG (150 kDa) and albumin (70 kDa), are 100–200-fold higher in the blood than in the CSF 51 . The present study demonstrated that even small molecules such as H 2 S (34 Da), which readily crosses the plasma membrane and is transported via the AE1 Cl - /HS - exchanger in erythrocytes 83, 84 , as well as H 2 S 2 (66 Da) and CysSSH (153 Da), showed significant differences in distribution between CSF and PLA in individuals with SZ, AD, and PD, suggesting the existence of some specific component(s) and regulatory mechanisms that govern the transport of these molecules across the BBB. Further studies are needed to identify the specific BBB component(s) whose dysfunction alters the transport of H 2 S, which are involved in the pathophysiology of these neurological and neurodegenerative diseases and are potential therapeutic targets. Methods Chemicals All methods were performed in accordance with the guidelines and regulations of chemical substance management and were approved by the Committees of Chemical Substance Management at Sanyo-Onoda City University and the National Center of Neurology and Psychiatry. Na 2 S 2 (Dojindo, Kumamoto, Japan), Na 2 S 3 (Dojindo), Na 2 S (Wako pure chemicals, Osaka, Japan), l-cysteine (Wako), and glutathione (Wako) were dissolved at 0.1 M in ultrapure water. These stock solutions were stored at −80 °C and used within a week. Ethical approval and study participants. Written informed consent was obtained from all participants. All experiments with human samples were approved by the Human Research Committee of Sanyo-Onoda City University (permission number: 23005), conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of NCNP, Japan (A2022-105 for Biobank, NCNP and A2024-072 for this specific study). Plasma samples were obtained from the NCNP Biobank, which is accredited under ISO 20387. Informed consent was obtained from all participants. Biobanking procedures were approved by the Institutional Review Board of the National Center of Neurology and Psychiatry (approval number: A2019-092), and each study was approved by the NCNP Biobank Utilization Review Committee. Blood was collected using VENOJECT ® II tubes (Terumo, Tokyo), centrifuged within 2 h of collection (2,500 × g , 10 min, 4 °C), and aliquoted into 96 Jacket tubes (FCR & Bio, Kobe). Samples were stored at −80 °C until further use. The participants included 20 patients with SZ, 10 with AD, 12 with PD, and 20 healthy controls. The mean age and sex ratios were matched across the three diagnostic groups. Participants with a history of central nervous system disease, severe head injury, or substance abuse were excluded. Patients were recruited from the National Center of Neurology and Psychiatry Hospital, Tokyo, Japan. Control subjects were recruited through advertisements in a free local magazine and our website announcement. All participants underwent a structured interview using the Mini-International Neuropsychiatric Interview (M.I.N.I.), Japanese version 85 , administered by trained psychologists or psychiatrists. For participants with schizophrenia and Major Depressive Disorder (MDD), a consensus diagnosis was made according to the DSM-IV criteria 86 based on the M.I.N.I., additional unstructured interviews, and information from medical records. Schizophrenic and depressive symptoms were assessed using the Japanese version of the Positive and Negative Syndrome Scale (PANSS) 87 and the Japanese version of GRID-Hamilton Depression Rating Scale (HAM-D) 88 , respectively. Medication status at the time of lumbar puncture was recorded. Daily doses of antipsychotics or antidepressants were converted to equivalent doses of chlorpromazine or imipramine, according to published guidelines 89 . Clinical data were managed in a database using the FileMaker server (FileMaker Inc., Santa Clara, CA, USA). AD dementia was diagnosed based on the criteria of the National Institute on Aging–Alzheimer’s Association (NIA-AA) criteria 90 . Diagnoses were established by board-certified psychiatrists or neurologists and verified using the clinical information recorded in the NCNP Biobank database. Patients with PD were diagnosed according to the Movement Disorder Society (MDS) clinical diagnostic criteria for PD 91 . Diagnoses were made by board-certified neurologists and confirmed through clinical records available in the NCNP Biobank. Note that the limitation of this study is the relatively small sample size, particularly when participants were subdivided by disease and smoking status. Preparation of CSF and PLA for LC-MS/MS. For LC-MS/MS analysis, H 2 S, H 2 S 2 , H 2 S 3 , CysSH, CysSSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in the CSF and PL samples were prepared according to a previously reported method with slight modifications 6, 92, 93 . Briefly, 2.8 ml of 50 mM monobromobimane (mBB) (Life Technologies, Carlsbad, CA, USA) was added to 70 ml CSF or PLA and incubated for 30 min in the dark at room temperature. Reaction was stopped by adding 5-sulfosalicyclic acid (SSA; final concentration, 2%) (Wako Pure Chemicals) and incubated for 10 min on ice. The reaction mixture was centrifuged at 12,000 × g for 10 min, and the supernatant was ultracentrifuged at 47,000 rpm for 60 min (Optima MAX-XP equipped with TLA55 rotor, Beckman Coulter, California, USA). The supernatant was analyzed using LC-MS/MS (Agilent 6470 Triple Quad LC/MS, Santa Clara, USA). LC-MS/MS analysis . Samples derivatized with mBB (Life Technologies) were analyzed using a triple-quadrupole mass spectrometer coupled to an HPLC (Agilent Technology, LC-MS/MS 6470) according to a previously reported method with slight modifications (Furuie et al., 2023). Samples were subjected to analysis on a reverse-phase Symmetry C18 HPLC column (3.5 mm, 2.1 × 150 mm, Waters, Milford, Massachusetts, USA) at a flow rate of 0.3 ml/min. The mobile phase consisted of (A) 0.1% formic acid (Wako Pure Chemicals, Osaka, Japan) in water and (B) 0.1% formic acid in methanol (Wako Pure Chemicals). Samples were separated by gradient elution technique: 0–7 min 5–90% B, 7–10 min 90% B, 10–10.1 min 90–5% B, and 10.1–15 min 5% B. The column oven temperature was maintained at 40 °C. The effluent was subjected to mass spectrometry using an electrospray ionization (ESI) interface operating in the positive-ion mode. The source temperature was set at 400 °C, and the ion spray voltage was set at 4.5 kV. Nitrogen was used as a nebulizer and drying gas. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor mass transitions in positive ion mode: CysS-mBB m/z 312.0→192.0, CysSS-mBB m/z 344.0→192.0, GS-mBB m/z 498.0→435.0, GSS-mBB m/z 530.0→192.0, GSSS-mBB m/z 562.0→192.0, mBB-S-mBB m/z 415.3→193, mBB-SS-mBB m/z 447.3→192, mBB-SSS-mBB m/z 479.3→192, Sulfite-mBB m/z 271.0→192.0, Thiosulfate-mBB m/z 303.0→192.0. Statistical analysis. All statistical analyses were performed using Microsoft Excel 2023 for Window 10 (Microsoft, Redmond, WA, USA) with the Analysis ToolPak add-in software. Differences between two groups were analyzed using Student’s t -test. Declarations Data availability All data included in this study are available in Supplementary Figures containing detailed statistical analyses. The data supporting the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Sciences, Sports, and Technology, Japan to H.K. (23K06016), and by Smoking Research Foundation to H.K. NCNP biobank is partly supported by a grant from Japan Agency for Medical Research and Development (AMED), GAPFREE4 (21ak0101151h0002) and by an Intramural Research Grant (6-1 and 6-7) for Neurological and Psychiatric Disorders of NCNP. Author Contributions Y.K. and H.K. performed experiments and data analysis, and H.K. supervised the study. Y.K., K.H., Y.O., T.T., C.F., Y.T., and H.K. wrote the manuscript with consultation from all authors. K.H., Y.O., T.T., C.F., and Y.T. interviewed participants and collected cerebrospinal fluid and plasma samples. M.T., and D.I. provided suggestions for LC-MS/MS experiments. T.N. suggested the study. Data availability statement All data generated during this study are included in this published article and supplementary information files. Competing interests The authors declare no competing interests. Additional information Supplementary information References Cirino, G., Szabo, C., Papapetropoulos, A. Physiological roles of hydrogen sulfide in mammalian cells, tissues, and organs. Physiol Rev. 103 , 31-276. (2023). Kumar, R., Banerjee, R. Regulation of the redox metabolome and thiol proteome by hydrogen sulfide. Crit Rev Biochem Mol Biol. 56 , 221-235. (2021). Kimura, H. 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06:29:55","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":178738,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7804547/v1/a4e2da49222dbb4efb9297ea.html"},{"id":94632511,"identity":"c2d54db8-9d37-47a4-be9e-58de3500d7ea","added_by":"auto","created_at":"2025-10-29 06:29:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":86817,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7804547/v1/ee8c1b889546e4feedb8235f.png"},{"id":94632512,"identity":"cdcd2302-b062-4e7d-b653-f4d5eadc9b2b","added_by":"auto","created_at":"2025-10-29 06:29:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":293777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndogenous levels of H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS, polysulfides and related molecules in the CSF and PLA of control young and old smokers and non-smokers.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e, Endogenous levels of H\u003csub\u003e2\u003c/sub\u003eS (\u003cstrong\u003ea\u003c/strong\u003e), CysSSH (\u003cstrong\u003eb\u003c/strong\u003e), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e (\u003cstrong\u003ec\u003c/strong\u003e), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e (\u003cstrong\u003ed\u003c/strong\u003e), CysSH (\u003cstrong\u003ee\u003c/strong\u003e), and GSH (\u003cstrong\u003ef\u003c/strong\u003e) in the CSF (\u003cstrong\u003eupper row\u003c/strong\u003e) and PLA (\u003cstrong\u003elower row\u003c/strong\u003e) obtained from control (Ct) young (37–55 years old) smokers (S) (n = 7) and non-smokers (nS) (n = 5), and those from control old (57–73 years old) smokers (n = 3) and non-smokers (n = 5) were compared. Both CSF and PLA were obtained from the same individuals. *p\u0026lt;0.05, **p\u0026lt;0.01 indicate significantly different, and \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.1 indicate tendency, Student \u003cem\u003et\u003c/em\u003e-test. All data are presented as mean \u003cu\u003e±\u003c/u\u003e SEM. \u003cstrong\u003eg\u003c/strong\u003e, Schematic representation of the changes in the levels of molecules in control young and old smokers and non-smokers.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7804547/v1/6b15fbb1e2977e8a57779a0c.png"},{"id":94640475,"identity":"93181092-f723-4be6-b59b-51801ab3c487","added_by":"auto","created_at":"2025-10-29 07:49:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":268982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndogenous levels of H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS, polysulfides, and related molecules in the CSF and PLA of patients with SZ compared with those of control young smokers and non-smokers.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e, Endogenous levels of H\u003csub\u003e2\u003c/sub\u003eS (\u003cstrong\u003ea\u003c/strong\u003e), CysSSH (\u003cstrong\u003eb\u003c/strong\u003e), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e (\u003cstrong\u003ec\u003c/strong\u003e), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e (\u003cstrong\u003ed\u003c/strong\u003e), CysSH (\u003cstrong\u003ee\u003c/strong\u003e), and GSH (\u003cstrong\u003ef\u003c/strong\u003e) in the CSF (\u003cstrong\u003eupper row\u003c/strong\u003e) and PLA (\u003cstrong\u003elower row\u003c/strong\u003e) obtained from SZ (35–56 years old) smokers (S) (n = 10) and non-smokers (nS) (n = 10) were compared those from control (Ct) young (37–55 years old) smokers (n = 7) and non-smokers (n = 5 ). Both CSF and PLA were obtained from the same individuals. *p\u0026lt;0.05, **p\u0026lt;0.01 indicate significantly different, and \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.1 indicate tendency, Student \u003cem\u003et\u003c/em\u003e-test. All data are presented as mean \u003cu\u003e±\u003c/u\u003e SEM. \u003cstrong\u003eg\u003c/strong\u003e, Schematic representation of the changes in the levels of molecules in SZ smokers and non-smokers.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7804547/v1/e66e3611bc5ac6fc8b2ec671.png"},{"id":94672243,"identity":"fdea1d31-945e-4f96-be17-92fe7bfd28e8","added_by":"auto","created_at":"2025-10-29 13:40:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":292692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndogenous levels of H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS, polysulfides, and related molecules in the CSF and PLA of patients with AD and PD compared with those of control old smokers and non-smokers.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e, Endogenous levels of H\u003csub\u003e2\u003c/sub\u003eS (\u003cstrong\u003ea\u003c/strong\u003e), CysSSH (\u003cstrong\u003eb\u003c/strong\u003e), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e (\u003cstrong\u003ec\u003c/strong\u003e), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e (\u003cstrong\u003ed\u003c/strong\u003e), CysSH (\u003cstrong\u003ee\u003c/strong\u003e), and GSH (\u003cstrong\u003ef\u003c/strong\u003e) in the CSF (\u003cstrong\u003eupper row\u003c/strong\u003e) and PLA (\u003cstrong\u003elower row\u003c/strong\u003e) obtained from AD (57–81 years old) smokers (S) (n = 5) and non-smokers (nS) (n = 5), were compared with those from PD (57–74 years old) smokers (n = 6) and non-smokers (n = 6) and control (Ct) old (59–74 years old) smokers (n = 3) and non-smokers (n = 5). Both CSF and PLA samples were obtained from the same individuals. *p\u0026lt;0.05, **p\u0026lt;0.01 indicate significantly different, and \u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.1 indicate tendency, Student \u003cem\u003et\u003c/em\u003e-test. All data are presented as mean \u003cu\u003e±\u003c/u\u003e SEM. \u003cstrong\u003eg\u003c/strong\u003e, Schematic representation of the changes in the levels of molecules in AD and PD smokers and non-smokers.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7804547/v1/9cfd7990c6e35aa931a3e78b.png"},{"id":94632524,"identity":"ba6c0979-3110-42bb-bcb9-fd7577ec6a87","added_by":"auto","created_at":"2025-10-29 06:29:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3598107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in localization of H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eS, polysulfides, and related molecules between CSF and PLA of patients with SZ, AD and PD compared with those of control individuals, and the effects of smoking habits. a\u003c/strong\u003e-\u003cstrong\u003ep\u003c/strong\u003e, Endogenous levels of H\u003csub\u003e2\u003c/sub\u003eS (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003eb\u003c/strong\u003e), CysSSH (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e (\u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e (\u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e), CysSH (\u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ej\u003c/strong\u003e), GSH (\u003cstrong\u003ek\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e), thiosulfate (TS) (\u003cstrong\u003em\u003c/strong\u003e, \u003cstrong\u003en\u003c/strong\u003e), and sulfite (\u003cstrong\u003eo\u003c/strong\u003e, \u003cstrong\u003ep\u003c/strong\u003e) in the CSF and PLA obtained from the same control (Ct) young (Y) (37–55 years old, \u003cstrong\u003efirst row\u003c/strong\u003e), SZ (35–56 years old, \u003cstrong\u003esecond row\u003c/strong\u003e), control old (O) (59–73 years old, \u003cstrong\u003ethird row\u003c/strong\u003e), AD (57–81 years old, \u003cstrong\u003eforth row\u003c/strong\u003e), PD (57–74 years old, \u003cstrong\u003efifth row\u003c/strong\u003e) non-smokers (nS) (\u003cstrong\u003ea, c, e, g, i, k, m, o\u003c/strong\u003e) and smokers (S) (\u003cstrong\u003eb, d, f, h, j, l, n, p\u003c/strong\u003e) were compared. The number of samples is indicated on the right side of each figure by a line corresponding to each color.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7804547/v1/2ce4a0920273f1f317b7552a.png"},{"id":97371397,"identity":"9eb0a9aa-91e5-48c7-998b-964e5ab483f3","added_by":"auto","created_at":"2025-12-03 16:28:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5642120,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7804547/v1/1a18982e-1030-4d04-8980-114b33b18af7.pdf"},{"id":94632515,"identity":"6c1b9ad8-ab7f-4c82-b8c8-ed15f3908731","added_by":"auto","created_at":"2025-10-29 06:29:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1490908,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"CSFPLANatCommunSupple.docx","url":"https://assets-eu.researchsquare.com/files/rs-7804547/v1/de78b33119af447d7a6c3e82.docx"},{"id":94632518,"identity":"4859a179-2ea6-40cf-9f47-a2d8f9269a67","added_by":"auto","created_at":"2025-10-29 06:29:54","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1665530,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"nrreportingsummarykimura.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7804547/v1/67e18beee17e50c1ec9ae882.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Hydrogen sulfide and polysulfide levels in cerebrospinal fluid and plasma of patients with schizophrenia, Alzheimer’s disease, Parkinson’s disease, and in relation to cigarette smoking","fulltext":[{"header":"Indroduction","content":"\u003cp\u003eHydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) is produced by enzymes, including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST), which also produce polysulfides H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003en\u003c/sub\u003e (n\u0026thinsp;\u003cspan type=\"DoubleUnderline\" class=\"DoubleUnderline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;2) and cysteine persulfide (CysSSH) \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Several other enzymes can also produce polysulfides, including sulfur quinone oxidoreductase (SQR), superoxide dismutase (SOD), myoglobin, catalase, myeloperoxidase, and cysteinyl tRNA synthetase (CARS) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. H\u003csub\u003e2\u003c/sub\u003eS is metabolized in the mitochondria by SQR and sulfur dioxygenase to sulfite, which is further metabolized by rhodanese to thiosulfate \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS and polysulfides exert various physiological effects \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In the nervous system, H\u003csub\u003e2\u003c/sub\u003eS and polysulfides facilitate the induction of hippocampal long-term potentiation, a synaptic model of memory formation, by enhancing the activity of N-methyl-D-aspartate (NMDA) receptors, facilitating the release of neurotransmitters such as glutamate, GABA, D-serine, a co-agonist of NMDA receptors, and activating transient receptor potential ankyrin 1 (TRPA1) channels in astrocytes, which regulate synaptic activity through the release of gliotransmitters \u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. H\u003csub\u003e2\u003c/sub\u003eS and polysulfides also protect neurons from oxidative stress called oxytosis, which has recently been proposed to be closely related to ferroptosis \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Disruptions in H\u003csub\u003e2\u003c/sub\u003eS/polysulfide production, as well as in the function of the cystine/glutamate antiporter (Xc\u003csup\u003e\u0026minus;\u003c/sup\u003e), a key target in oxytosis and ferroptosis, have been implicated in the pathogenesis of Alzheimer\u0026rsquo;s disease (AD), Parkinson\u0026rsquo;s disease (PD), and other neurological disorders, including schizophrenia (SZ) \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Polysulfides release Nrf2 from the Keap1/Nrf2 complex to upregulate antioxidant genes and protect cells from oxidative stress \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eLow concentrations of H\u003csub\u003e2\u003c/sub\u003eS exhibit beneficial effects, whereas high concentrations are toxic. H\u003csub\u003e2\u003c/sub\u003eS levels are increased by its overproduction or deficiency of its metabolizing enzymes. For example, CBS, encoded on chromosome 21, which is present in triplicate in individuals with Down\u0026rsquo;s syndrome, is overexpressed, leading to excessive H\u003csub\u003e2\u003c/sub\u003eS production that inhibits mitochondrial electron transport, oxygen consumption, and ATP production \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Another example is sulfur dioxygenase, an H\u003csub\u003e2\u003c/sub\u003eS metabolizing enzyme, which is deficient in ethylmalonic encephalopathy, leading to excessive H\u003csub\u003e2\u003c/sub\u003eS levels that suppress cytochrome c oxidase in the muscle and brain \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSZ is a chronic and severe mental disorder that affects thoughts, feelings, and behaviors. The symptoms fall into three categories: positive, negative, and cognitive. Both excess and deficiency of H\u003csub\u003e2\u003c/sub\u003eS and polysulfides have been proposed to be involved in the pathogenesis of SZ. H\u003csub\u003e2\u003c/sub\u003eS levels in the PLA were significantly lower in patients with SZ than in normal individuals, and a positive association was observed between H\u003csub\u003e2\u003c/sub\u003eS levels in the PLA and working and visual memory \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Thiol homeostasis was disrupted in such patients due to a shift from sulfide (reduced) to disulfide bond formation (oxidized) \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e In contrast, 3MST levels in SZ were positively correlated with symptom severity scores \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAD is a chronic neurodegenerative disease that affects cognitive function and memory. The cause of the disease is mostly sporadic, with fewer familial diseases. H\u003csub\u003e2\u003c/sub\u003eS suppressed the activities of β-secretase and γ-secretase to prevent the production of amyloid β-protein via activation of the PI3K/Akt pathway in amyloid precursor/presenilin 1 transgenic mice \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. H\u003csub\u003e2\u003c/sub\u003eS suppressed tau hyperphosphorylation, which generated neurofibrillary tangles by inhibiting glycogen synthase kinase 3β (GSK3β) \u003csup\u003e19\u003c/sup\u003e. CSE levels were decreased in postmortem human brains and AD mouse models with TauP301L, an amyloid precursor protein with a Swedish mutation, and presenilin 1M146V \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Another AD mouse model with five familial AD mutations, in which there was no significant difference in the expression levels of CBS, CSE, CARS1, and CARS2, showed lower levels of total polysulfides and protein polysulfides in the brain than in the wild-type mouse brain; however, no significant differences in the levels of GSH, GSSH, and H\u003csub\u003e2\u003c/sub\u003eS were observed \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePD is a neurodegenerative disorder that causes motor dysfunctions. Parkin and α-synuclein are associated with pathophysiology of PD. Parkin is an E3 ubiquitin ligase that ubiquitinates diverse substrates and is responsible for the clearance of misfolded proteins, including α-synuclein. Parkin activity was suppressed by S-nitrosylation, thereby decreasing its protective effect in the brains of patients with PD \u003csup\u003e21\u003c/sup\u003e. In contrast, parkin existed in active and persulfidated (S-sulfhydrated) state in the brains of healthy individuals \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. H\u003csub\u003e2\u003c/sub\u003eS attenuated neuronal apoptosis in the substantia nigra in a rat model of PD \u003csup\u003e23\u003c/sup\u003e. The intestinal microbiota is involved in neuroinflammation in PD, and H\u003csub\u003e2\u003c/sub\u003eS producing gut bacterium \u003cem\u003eDesulfovibrio\u003c/em\u003e is significantly increased in PD compared to that in normal individuals and is positively correlated with disease severity \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eCigarette smoke contains H\u003csub\u003e2\u003c/sub\u003eS, polysulfides, and related sulfur-containing molecules \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Smoke produced by one cigarette contains 31.6 \u0026micro;g (0.93 \u0026micro;mol) H\u003csub\u003e2\u003c/sub\u003eS, 40.7 \u0026micro;g (0.68 \u0026micro;mol) carbonyl sulfide, which converts to H\u003csub\u003e2\u003c/sub\u003eS and CO\u003csub\u003e2\u003c/sub\u003e in the presence of water, and 17.6 \u0026micro;g (0.19 \u0026micro;mol) methyl disulfide. Considering that the endogenous concentrations in the brain are in the range of 14 nM\u0026ndash;3 \u0026micro;M for H\u003csub\u003e2\u003c/sub\u003eS and 0.2 \u0026micro;M for H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, the acute effect of cigarette smoke is indispensable \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Although H\u003csub\u003e2\u003c/sub\u003eS and polysulfides inhaled from cigarette smoke may be immediately metabolized and may not remain in the body, smoking habits exert various effects on SZ, AD, and PD by altering the endogenous production and metabolism of these molecules.\u003c/p\u003e\u003cp\u003eSZ is frequently comorbid with smoking behaviors. Case-control and cohort studies have shown that smoking behaviors cause significantly higher risk of SZ compared with that in non-smokers \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Meta-analyses have also shown that smokers have a relatively higher risk of developing SZ than that of non-smokers \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Even a causal role of smoking in the development of SZ has been proposed \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. An increased prevalence of cigarette smoking is characteristic of individuals with SZ \u003csup\u003e32\u003c/sup\u003e. The microbiome, including bacteria and bacteriophages, influences SZ through neurological and immunological mechanisms. Bacteriophage levels are also influenced by cigarette smoking \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEpidemiological evidence from meta-analyses and cohort studies indicates that smoking is associated with a significantly increased risk of AD pathology \u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, while some prospective cohort studies and Mendelian randomization analyses have shown no association between smoking and AD neuropathology \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Smoking potentially affects the diversity, composition, and abundance of the microbiota and increases anaerobic bacteria and pathogens, leading to the onset and progression of AD \u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn contrast to AD and SZ, smoking has been shown to be associated with a decreased risk of PD \u003csup\u003e40, 41\u003c/sup\u003e. Although PD smokers are more likely to die from smoking-related cancers than PD non-smokers, they tend to have fewer deaths from neurological causes \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Nicotine is the dominant candidate underlying the reduced risk of PD; however, randomized controlled clinical trials of nicotine in PD have failed to demonstrate its benefit on motor function \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Recent findings suggest that non-nicotine components in cigarette smoke may contribute to this protective effect \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Cigarette smoke can alter the composition of microbiota to mitigate intestinal inflammation, leading to reduced misfolding of a-synclein in the enteric nervous system \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe blood-brain barrier (BBB) separates neuronal environment from peripheral blood. The molecular size threshold for passive diffusion across the BBB is 400\u0026ndash;600 Da, and lipophilicity determines the effectiveness of passive diffusion across the BBB \u003csup\u003e\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. An early pathological hallmark of AD is BBB dysfunction, which is characterized by barrier leakage \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Accumulating evidence has revealed BBB dysfunction in patients with PD and SZ \u003csup\u003e52, 53\u003c/sup\u003e. The present study examined the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, CysSSH, cysteine (CysSH), GSH, GSH persulfide (GSSH), GSH trisulfide (GSSSH), thiosulfate, and sulfite in both CSF and PLA obtained from the same individuals. Even the largest molecule examined in this study, GSH (MW: 307) and its persulfidated counterparts, can pass through the BBB, but the CSF to PLA ratio of some of these molecules differed significantly in individuals with SZ, AD, and PD compared with healthy controls. The effects of smoking behaviors differed significantly among these diseases.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTo examine the pathophysiological roles of H\u003csub\u003e2\u003c/sub\u003eS, polysulfides, and related molecules in SZ, AD, and PD, as well as the effects of smoking habits in these neurological diseases, the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, CysSH, CysSSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in the CSF and PLA obtained from the same individuals were examined. The baseline characteristics of the study participants and the type of diseases are shown in Table 1, and the age distribution of the participants is shown in Fig. 1. Individuals 35\u0026ndash;56 years old were grouped as young individuals and 57\u0026ndash;81 years old as old individuals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince it has been reported that aged cells contain fewer persulfidated molecules due to the loss of H\u003csub\u003e2\u003c/sub\u003eS producing enzymes, including CSE and 3MST \u003csup\u003e54\u003c/sup\u003e, the levels of H\u003csub\u003e2\u003c/sub\u003eS, polysulfides, and related molecules in the CSF and PLA of control young non-smokers were compared to those of their older counterparts. The levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, CysSSH, CysSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in both CSF or PLA were not significantly different between control young and old non-smokers (Fig. 2 and Supplementary Fig. S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe effects of smoking habits on the levels of these molecules in control young and old smokers were examined by comparing them with those in corresponding control non-smokers. CysSSH levels in the CSF of control old smokers were approximately 0.5 fold of those of control old non-smokers and approximately 0.6 fold of those of control young smokers (Fig. 2b, g), while GSH and CysSH levels were approximately 2.2- and 1.7-fold higher in control old smokers than in control young smokers (Fig. 2e-g). In contrast, the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH in the PLA of control old smokers were approximately 4.4-, 8.2-, and 5.5-fold, respectively, higher than those in the corresponding non-smokers, and H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e and CysSSH levels were approximately 3.3- and 2.1-fold higher than those in control young smokers (Fig. 2a-c, g). These observations suggest that smoking habits have a greater influence on old individuals rather than on young individuals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsidering these observations and the age distribution of donors, data obtained from patients with SZ were compared with those of control young individuals, whereas those from patients with AD and PD were compared with those of control old individuals. Data from two patients with SZ older than 57 years (age of 60 and 65 years) and one patient with PD younger than 56 years (age of 52 years) were not included in the present analysis because the ages of these individuals deviated from the defined range (Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eS and polysulfides in SZ\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe levels of H\u003csub\u003e2\u003c/sub\u003eS, polysulfides, and related molecules in the CSF and PLA of patients with SZ were compared with those of control young individuals. The levels of H\u003csub\u003e2\u003c/sub\u003eS (~0.6 fold), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e (~0.4 fold), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e (~0.5 fold), and CysSSH (~0.6 fold) were lower in the CSF of SZ non-smokers than in those of control young non-smokers, while there were no significant differences in the PLA levels (Fig. 3a-d). No significant differences in the levels of CysSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in both CSF and PLA of SZ non-smokers were observed compared with those of control young non-smokers (Fig. 3e, f, and Supplementary Fig. S3). Since the CSF is the extracellular environment of neurons and glia in the brain, these observations suggest that decreased levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, and CysSSH in the CSF are associated with the pathophysiology of SZ.\u003c/p\u003e\n\u003cp\u003eThe effects of smoking habits on the levels of H\u003csub\u003e2\u003c/sub\u003eS, polysulfides, and related molecules in SZ were examined. Although CysSSH levels in the CSF of SZ non-smokers were significantly lower (~0.6 fold) than those of control young non-smokers as described previously (Fig. 3b), those of SZ smokers were almost similar to those of control young non-smokers (~0.9 fold) (Fig. 3b). H\u003csub\u003e2\u003c/sub\u003eS levels in the CSF of SZ smokers showed a similar result (from ~0.6 to ~0.7) (Fig. 3a). The recovery of H\u003csub\u003e2\u003c/sub\u003eS and CysSSH levels in the CSF by smoking habits to almost those of control individuals may be related to the smoking preference of patients with SZ (Fig. 3g).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to the CSF, the levels of H\u003csub\u003e2\u003c/sub\u003eS (~3.5 fold), H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e (~2.8), and CysSSH (~3.4) in the PLA of SZ smokers were significantly higher than those of SZ non-smokers and control young non-smokers (Fig. 3a-c). These observations indicate that the levels of H\u003csub\u003e2\u003c/sub\u003eS and polysulfides are differently regulated in the CSF and PLA, and that smoking habits influence this regulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eS and polysulfides in AD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChanges in the levels of H\u003csub\u003e2\u003c/sub\u003eS, polysulfides, and related molecules in the CSF and PLA of patients with AD were compared with those of control old individuals. The levels of H\u003csub\u003e2\u003c/sub\u003eS (~0.7 fold) and CysSSH (~0.6) in the CSF of AD non-smokers were lower than those of control old non-smokers, while no significant differences were observed in the PLA levels (Fig. 4a, b). These observations suggest that decrease in both molecules in the CSF may be related to the pathophysiology of AD, similar to that of SZ. There were no significant differences in the levels of CysSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in both CSF and PLA of patients with AD compared with control old individuals (Fig. 4e, f, Supplementary Fig. S4).\u003c/p\u003e\n\u003cp\u003eSmoking habits had no significant effect on the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH in the CSF of patients with AD, while these levels were approximately 2.2-, 2.6-, and 2.1-fold, respectively, higher in the PLA of AD smokers than those in AD non-smokers and control old non-smokers (Fig. 4a-c). Although the effect of smoking habits on the CSF of patients with AD was different from that of patients with SZ, the effect on the PLA was similar between AD and SZ (Fig. 3g and 4g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eS and polysulfides in PD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe levels of H\u003csub\u003e2\u003c/sub\u003eS, polysulfides, and related molecules were measured in the CSF and PLA of patients with PD and compared with those of control old individuals. No significant differences in the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH were observed in the CSF, while the PLA levels of these molecules were significantly higher in PD non-smokers than in control old individuals (approximately 3.4-, 4.8-, and 3.1-fold, respectively) (Fig. 4a-c). These observations suggest that peripheral inflammation and intestinal microbiota may be involved in the pathophysiology of PD. Changes in these molecules in the CSF and PLA of patients with PD were strikingly different from those in SZ and AD (Fig. 3g and 4g).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe association between smoking habits and a reduced risk of PD is among the most robust environmental and lifestyle correlations observed in neuroepidemiology \u003csup\u003e45\u003c/sup\u003e. Smoking habits significantly increased the levels of H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e in the CSF (~1.5 fold) compared with PD non-smokers, control old smokers, and control non-smokers (Fig. 4d). In contrast, in the PLA, the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH in PD non-smokers (approximately 3.4-, 4.8-, and 3.1-fold, respectively) were higher than those in control old non-smokers, and these were decreased by smoking habits to approximately 1.5-, 1.1-, and 1.4-fold, respectively, of control old non-smokers (Fig. 4a-c, g), suggesting an association between smoking habits and a reduced risk of PD. The effects of smoking on these molecules in PD were significantly different from those observed in SZ and AD (Fig. 3g and 4g).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u003csub\u003e2\u003c/sub\u003eS and polysulfides distribution between CSF and PLA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe BBB separates the neuronal environment from the peripheral blood and plays a crucial role in maintaining homeostasis of the central nervous system. BBB dysfunction has been reported in patients with SZ, AD, and PD \u003csup\u003e51-53\u003c/sup\u003e. To understand the specificity of the BBB to H\u003csub\u003e2\u003c/sub\u003eS, polysulfides, and related molecules in these diseases, the distribution of these molecules between the CSF and PLA obtained from the same individuals was examined. The levels of H\u003csub\u003e2\u003c/sub\u003eS, CysSH, and CysSSH (as shown by the slopes of the lines) were higher in the PLA than in the CSF (Fig. 5a-d, i, j), whereas those of GSH and sulfite were lower in the PLA (Fig. 5k, l, o, p). H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, thiosulfate, GSSH, and GSSSH were almost equally distributed between the CSF and PLA (Fig. 5e-h, m, n, and Supplementary Fig. S5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe distribution of H\u003csub\u003e2\u003c/sub\u003eS and CysSSH between the PLA and CSF were not significantly different in SZ non-smokers compared with control Young non-smokers (Fig. 5a, c, and Supplementary Fig. S7a, b). H\u003csub\u003e2\u003c/sub\u003eS and CysSSH were more inclined to PLA (approximately 1.5- and 2.1-fold, respectively) in AD non-smokers than in control old non-smokers (Fig. 5a, c, and Supplementary Fig. S8a, b). In contrast, the localization of H\u003csub\u003e2\u003c/sub\u003eS and CysSSH was significantly more inclined to PLA than to CSF (approximately 3.4- and 4.2-fold, respectively) in PD non-smokers than in control old non-smokers (Fig. 5a, c, and Supplementary Fig. S8a, b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSmoking habits affect older individuals more than their younger counterparts. The PLA/CSF ratios of H\u003csub\u003e2\u003c/sub\u003eS and CysSSH in control young smokers were approximately 2.3- and 2.0-fold of those in control young non-smokers, while those in control old smokers were approximately 5.7- and 14.0-fold of those in control old non-smokers (Fig. 5a-d, and Supplementary Fig. S6a, b). Smoking habits induced shifts in the localization of H\u003csub\u003e2\u003c/sub\u003eS and CysSSH from the CSF to the PLA in SZ (PLA/CSF: approximately 3- and 2.5-fold, respectively) (Supplementary Fig. S7a, b). In contrast, the localization of both molecules shifted less to PLA due to smoking habits in PD (PLA/CSF: approximately 0.4-fold each), which almost recovered to the PLA/CSF ratio of control old non-smokers (Fig. 5a-d and Supplementary Fig. S8a, b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese observations suggest that not only the levels of H\u003csub\u003e2\u003c/sub\u003eS and polysulfides but also their localization between the CSF and PLA have specific inclinations in SZ, AD, and PD. The difference in distribution may be caused by changes in BBB function and peripheral inflammation, as well as a result of smoking habits.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study showed that the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH are distinctively altered in SZ, AD, and PD and are affected by smoking habits. In SZ and AD, the levels of H\u003csub\u003e2\u003c/sub\u003eS and CysSSH significantly decreased in the CSF but not in the PLA, compared with those in the control group (Figs. 3, 4). In contrast, in PD, the levels of these molecules were significantly increased only in the PLA.\u0026nbsp;The effects of smoking habits were divided into two groups: SZ and AD vs. PD. Smoking habits changed the endogenous levels of these molecules and their distribution between the CSF and PLA, suggesting a potential change in BBB function and involvement of peripheral inflammation.\u003c/p\u003e\n\u003cp\u003eThe levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, and CysSSH in the CSF of individuals with SZ were significantly lower than those of control individuals (Fig. 3a-d). As the CSF is the extracellular environment of neurons and glia in the brain, changes in the levels of H\u003csub\u003e2\u003c/sub\u003eS and polysulfides in the CSF may directly affect the activity of neurons, and therefore, associated with the pathophysiology of diseases of the central nervous system.\u0026nbsp;Modulation of\u0026nbsp;the cystine/glutamate antiporter\u0026nbsp;Xc\u003csup\u003e-\u003c/sup\u003e expression has been associated with many neurological and psychiatric disorders, including\u0026nbsp;SZ, AD,\u0026nbsp;and PD \u003csup\u003e55, 56\u003c/sup\u003e. Xc\u003csup\u003e-\u003c/sup\u003e imports cystine,\u0026nbsp;which is reduced to\u0026nbsp;CysSH\u0026nbsp;in cells, while exporting glutamate from the cell. The hypoglutamatergic hypothesis concerns suppression of the activity of Xc\u003csup\u003e-\u003c/sup\u003e as a contributory factor to the development of SZ \u003csup\u003e57\u003c/sup\u003e.\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eS and polysulfides enhance the activity of Xc\u003csup\u003e-\u003c/sup\u003e \u003csup\u003e8, 58, 59\u003c/sup\u003e. These molecules also regulate the release of neurotransmitters such as GABA, glutamate, and D-serine. Abnormalities in the balance between glutamatergic and GABAergic neuronal activities have been implicated in\u0026nbsp;the pathophysiology of SZ \u003csup\u003e6, 60\u003c/sup\u003e. 3MST-knockout rats, which have lower levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, and CysSSH in the brain, exhibit SZ-like hyperlocomotion following the administration of MK-801,\u0026nbsp;a non-specific NMDA receptor antagonist, compared with wild-type rats \u003csup\u003e6\u003c/sup\u003e. These observations suggest that decreased levels of H\u003csub\u003e2\u003c/sub\u003eS and polysulfides in\u0026nbsp;the CSF\u0026nbsp;are likely involved in\u0026nbsp;the pathophysiology of SZ.\u003c/p\u003e\n\u003cp\u003eThe levels of H\u003csub\u003e2\u003c/sub\u003eS and CysSSH in patients with AD were significantly lower in the CSF than in those of control individuals, whereas no significant differences were observed in the levels of these molecules in the PLA, similar to SZ (Fig. 4a, b, g). H\u003csub\u003e2\u003c/sub\u003eS and polysulfides protect neurons from oxidative stress and regulate neuronal transmission \u003csup\u003e6, 8, 58, 59\u003c/sup\u003e. The levels of CSE and persulfidation, which add sulfur to the thiol of cysteine residues, were decreased in postmortem brains of patients with AD. H\u003csub\u003e2\u003c/sub\u003eS produced by CSE prevented pathological phosphorylation of tau by persulfidating GSK3b\u0026nbsp;and inhibited its catalytic activity in\u0026nbsp;an\u0026nbsp;AD mouse model \u003csup\u003e19\u003c/sup\u003e. These observations suggest that a lack of H\u003csub\u003e2\u003c/sub\u003eS and CysSSH in\u0026nbsp;the CSF\u0026nbsp;is involved in the pathophysiology of AD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn PD, the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH in the PLA were significantly higher than those in control individuals (approximately 3\u0026ndash;5 fold), while no significant difference were observed in the CSF levels (Fig. 4a-c, g). This is a critical difference in the distribution of H\u003csub\u003e2\u003c/sub\u003eS and CysSSH in PD compared with that in SZ and AD, where the levels of these molecules decreased in the CSF but did not change in the PLA (Fig. 3 and 4). PD pathophysiology has an influence on regions outside the brain, including peripheral blood \u003csup\u003e61\u003c/sup\u003e. Circulatory system may contribute to PD pathogenesis through the spread of\u0026nbsp;a-synuclein, and BBB allows\u0026nbsp;a-synuclein to infiltrate in the brain \u003csup\u003e62\u003c/sup\u003e. More than 99% of\u0026nbsp;a-synuclein is present in erythrocytes in peripheral blood, and erythrocytes express 3MST, which can produce H\u003csub\u003e2\u003c/sub\u003eS and polysulfides \u003csup\u003e63-65\u003c/sup\u003e. H\u003csub\u003e2\u003c/sub\u003eS/polysulfides persulfidate and activate parkin, which is also expressed in peripheral blood lymphocytes, thereby promoting the clearance of misfolded proteins, including\u0026nbsp;a-synuclein \u003csup\u003e22\u003c/sup\u003e. The intestinal microbiota is involved in neuroinflammation in PD \u003csup\u003e25\u003c/sup\u003e. Numerous pro-inflammatory cytokines, such as IL-6, TNF, and IL-2, are increased in the blood of\u0026nbsp;patients\u0026nbsp;with PD \u003csup\u003e66\u003c/sup\u003e, and PD blood\u0026nbsp;monocytes have a higher proliferative capacity than those of control individuals \u003csup\u003e67\u003c/sup\u003e. The levels of H\u003csub\u003e2\u003c/sub\u003eS and polysulfides, which can mitigate inflammatory stress and enhance the activity of Xc- interacting with blood in\u0026nbsp;the BBB to\u0026nbsp;protect neurons at physiological concentrations, are increased to unfavorable levels in\u0026nbsp;the PLA of patients with PD\u0026nbsp;\u003csup\u003e8, 68, 69\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe effects of smoking habits were considerably non-consistent between patients with SZ and AD, and those with PD. SZ is reportedly associated with smoking behaviors. Meta-analyses have shown evidence of an increased relative risk of SZ in smokers compared with that in non-smokers \u003csup\u003e70\u003c/sup\u003e. The present study shows that the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH in the CSF decreased in SZ and were increased with smoking habits to levels that were not significantly different from those of control non-smokers (Fig. 3a-c, g), suggesting that it may be related to the preference of patients with SZ to smoking. In SZ non-smokers, the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH in the PLA, which were not significantly different from those of control individuals, increased with smoking habits to levels more than two times those of control non-smokers (Fig. 3a-c, g). Chronic peripheral inflammation is associated with the pathophysiology of SZ and has been reported to be associated with smoking preference \u003csup\u003e71\u003c/sup\u003e. The levels of H\u003csub\u003e2\u003c/sub\u003eS and polysulfides increased to suppress peripheral inflammation but reached unfavorable levels.\u003c/p\u003e\n\u003cp\u003eMeta-analyses and cohort-based studies have indicated that smoking is associated with a significantly increased risk of AD neuropathology and associated dementia \u003csup\u003e36, 72, 73\u003c/sup\u003e. The present study showed that the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH in the CSF were not significantly altered by smoking habits, while those in the PLA were increased by more than two times those of control non-smoking individuals, similar to SZ (Fig. 4a-c, g, and 3g).\u0026nbsp;Smoking potentially affects the diversity, composition, and abundance of microbiota, leading to the onset and progression of AD \u003csup\u003e39\u003c/sup\u003e.\u0026nbsp;Smoking also activates immune cells and induces vascular inflammation, including leukocyte infiltration,\u0026nbsp;in AD \u003csup\u003e74, 75\u003c/sup\u003e. Smoking habits have been associated with vascular endothelial dysfunction and chronic peripheral inflammation in AD and SZ by increasing IL-1b\u0026nbsp;and TNFa\u0026nbsp;\u003csup\u003e71, 76, 77\u003c/sup\u003e. Although H\u003csub\u003e2\u003c/sub\u003eS and polysulfides suppress inflammatory responses and exert cytoprotective effects against oxidative stress by activating Xc\u003csup\u003e-\u003c/sup\u003e and Nrf2 \u003csup\u003e8, 12\u003c/sup\u003e, the levels of these molecules increase to unfavorable levels in AD, similar\u0026nbsp;to\u0026nbsp;SZ.\u003c/p\u003e\n\u003cp\u003eIn contrast to SZ and AD, smoking has been reported to be associated with a reduced risk of developing PD \u003csup\u003e46\u003c/sup\u003e. As randomized controlled clinical trials of nicotine in patients with PD have failed to demonstrate a benefit for motor function \u003csup\u003e43\u003c/sup\u003e, non-nicotine components in cigarette smoke have been suggested to contribute to this protective effect \u003csup\u003e46\u003c/sup\u003e. H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e levels were significantly increased (~1.5 fold) in the CSF of smokers with PD (Fig. 4d, g).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ePolysulfides activate the Nrf2 pathway and Xc\u003csup\u003e-\u003c/sup\u003e to protect cells from oxidative stress \u003csup\u003e8, 12\u003c/sup\u003e. High concentrations of polysulfides and H\u003csub\u003e2\u003c/sub\u003eS inhibit the activity of monoamine oxidase, which degrades dopamine, to effectively preserve dopamine in the brain \u003csup\u003e78, 79\u003c/sup\u003e. In contrast to SZ and AD, smoking habits decreased the levels of H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, and CysSSH in the PLA of patients with PD to less than half of those in PD non-smokers and almost equal to the levels in control non-smokers (Fig. 4a-c, g). Cigarette smoke can modify the composition of microbiota to mitigate intestinal inflammation, thereby reducing misfolding of\u0026nbsp;a-synclein in the enteric nervous system \u003csup\u003e47\u003c/sup\u003e. Smoking habits also alter\u0026nbsp;the physicochemical properties of erythrocyte membranes, which may decrease the levels of\u0026nbsp;a-synuclein in\u0026nbsp;the blood\u0026nbsp;and overexpression of H\u003csub\u003e2\u003c/sub\u003eS and polysulfides in PD \u003csup\u003e64, 80\u003c/sup\u003e. These effects of smoking may be related to its observed association with a reduced\u0026nbsp;risk\u0026nbsp;of PD.\u003c/p\u003e\n\u003cp\u003eEmerging evidence indicates that BBB dysfunction is a key pathophysiological factor associated with tight junction abnormalities and endothelial dysfunction in SZ \u003csup\u003e53\u003c/sup\u003e. Recent clinical data have emphasized the association between BBB leakage and cognitive decline in patients with AD \u003csup\u003e51, 81\u003c/sup\u003e, and BBB alterations in PD \u003csup\u003e82\u003c/sup\u003e. The most commonly used clinical parameter to characterize barrier leakage is the CSF-to-serum ratio. In healthy individuals, the levels of blood-borne proteins, such as IgG (150 kDa) and albumin (70 kDa), are 100\u0026ndash;200-fold higher in the blood than in the CSF \u003csup\u003e51\u003c/sup\u003e. The present study demonstrated that even small molecules such as H\u003csub\u003e2\u003c/sub\u003eS (34 Da), which readily crosses the plasma membrane and is transported via the AE1 Cl\u003csup\u003e-\u003c/sup\u003e/HS\u003csup\u003e-\u003c/sup\u003e exchanger in erythrocytes \u003csup\u003e83, 84\u003c/sup\u003e, as well as H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e (66 Da) and CysSSH (153 Da), showed significant differences in distribution between CSF and PLA in individuals with SZ, AD, and PD, suggesting the existence of some specific component(s) and regulatory mechanisms that govern the transport of these molecules across the BBB. Further studies are needed to identify the specific BBB component(s) whose dysfunction alters the transport of H\u003csub\u003e2\u003c/sub\u003eS, which are involved in the pathophysiology of these neurological and neurodegenerative diseases and are potential therapeutic targets.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eChemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll methods were performed in accordance with the guidelines and regulations of chemical substance management and were approved by the Committees of Chemical Substance Management at Sanyo-Onoda City University and the National Center of Neurology and Psychiatry. Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e (Dojindo, Kumamoto, Japan), Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e (Dojindo), Na\u003csub\u003e2\u003c/sub\u003eS (Wako pure chemicals, Osaka, Japan), l-cysteine (Wako), and glutathione (Wako) were dissolved at 0.1 M in ultrapure water. These stock solutions were stored at\u0026thinsp;\u0026minus;80 \u0026deg;C and used within a week.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and study participants.\u003c/strong\u003e Written informed consent was obtained from all participants. All experiments with human samples were approved by the Human Research Committee of Sanyo-Onoda City University (permission number: 23005), conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of NCNP, Japan (A2022-105 for Biobank, NCNP and A2024-072 for this specific study). Plasma samples were obtained from the NCNP Biobank, which is accredited under ISO 20387. Informed consent was obtained from all participants. Biobanking procedures were approved by the Institutional Review Board of the National Center of Neurology and Psychiatry (approval number: A2019-092), and each study was approved by the NCNP Biobank Utilization Review Committee. Blood was collected using VENOJECT\u003csup\u003e\u0026reg;\u003c/sup\u003e II tubes (Terumo, Tokyo), centrifuged within 2 h of collection (2,500 \u0026times; \u003cem\u003eg\u003c/em\u003e, 10 min, 4 \u0026deg;C), and aliquoted into 96 Jacket tubes (FCR \u0026amp; Bio, Kobe). Samples were stored at \u0026minus;80 \u0026deg;C until further use.\u003c/p\u003e\n\u003cp\u003eThe participants included 20 patients with SZ, 10 with AD, 12 with PD, and 20 healthy controls. The mean age and sex ratios were matched across the three diagnostic groups. Participants\u0026nbsp;with a history of central nervous system disease, severe head injury, or substance abuse were excluded. Patients were recruited from the National Center of Neurology and Psychiatry Hospital, Tokyo, Japan. Control subjects were recruited through advertisements in a free local magazine and our website announcement.\u003c/p\u003e\n\u003cp\u003eAll participants underwent a structured interview using the Mini-International Neuropsychiatric Interview (M.I.N.I.), Japanese version \u003csup\u003e85\u003c/sup\u003e, administered by trained psychologists or psychiatrists. For participants with schizophrenia and Major Depressive Disorder (MDD), a consensus diagnosis was made according to the DSM-IV criteria \u003csup\u003e86\u003c/sup\u003e based on the M.I.N.I., additional unstructured interviews, and information from medical records. Schizophrenic and depressive symptoms were assessed using the Japanese version of the Positive and Negative Syndrome Scale (PANSS) \u003csup\u003e87\u003c/sup\u003e and the Japanese version of GRID-Hamilton Depression Rating Scale (HAM-D) \u003csup\u003e88\u003c/sup\u003e, respectively. Medication status at the time of lumbar puncture was recorded. Daily doses of antipsychotics or antidepressants were converted to equivalent doses of chlorpromazine or imipramine, according to published guidelines \u003csup\u003e89\u003c/sup\u003e. Clinical data were managed in a database using the FileMaker server (FileMaker Inc., Santa Clara, CA, USA).\u003c/p\u003e\n\u003cp\u003eAD dementia was diagnosed based on the criteria of the National Institute on Aging\u0026ndash;Alzheimer\u0026rsquo;s Association (NIA-AA) criteria \u003csup\u003e90\u003c/sup\u003e. Diagnoses were established by board-certified psychiatrists or neurologists and verified using the clinical information recorded in the NCNP Biobank database.\u003c/p\u003e\n\u003cp\u003ePatients with PD were diagnosed according to the Movement Disorder Society (MDS) clinical diagnostic criteria for PD \u003csup\u003e91\u003c/sup\u003e. Diagnoses were made by board-certified neurologists and confirmed through clinical records available in the NCNP Biobank.\u003c/p\u003e\n\u003cp\u003eNote that the limitation of this study is the relatively small sample size, particularly when participants were subdivided by disease and smoking status.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of CSF and PLA for LC-MS/MS.\u0026nbsp;\u003c/strong\u003eFor LC-MS/MS analysis, H\u003csub\u003e2\u003c/sub\u003eS, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e, H\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e3\u003c/sub\u003e, CysSH, CysSSH, GSH, GSSH, GSSSH, thiosulfate, and sulfite in the CSF and PL samples were prepared according to a previously reported method with slight modifications \u003csup\u003e6, 92, 93\u003c/sup\u003e. Briefly, 2.8 ml of 50 mM monobromobimane (mBB) (Life Technologies, Carlsbad, CA, USA) was added to 70 ml CSF or PLA and incubated for 30 min in the dark at room temperature. Reaction was stopped by adding 5-sulfosalicyclic acid (SSA; final concentration, 2%) (Wako Pure Chemicals) and incubated for 10 min on ice. The reaction mixture was centrifuged at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min, and the supernatant was ultracentrifuged at 47,000 rpm for 60 min (Optima MAX-XP equipped with TLA55 rotor, Beckman Coulter, California, USA). The supernatant was analyzed using LC-MS/MS (Agilent 6470 Triple Quad LC/MS, Santa Clara, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS/MS analysis\u003c/strong\u003e. Samples derivatized with mBB (Life Technologies) were analyzed using a triple-quadrupole mass spectrometer coupled to an HPLC (Agilent Technology, LC-MS/MS 6470) according to a previously reported method with slight modifications (Furuie et al., 2023). Samples were subjected to analysis on a reverse-phase Symmetry C18 HPLC column (3.5 mm, 2.1 \u0026times; 150 mm, Waters, Milford, Massachusetts, USA) at a flow rate of 0.3 ml/min. The mobile phase consisted of (A) 0.1% formic acid (Wako Pure Chemicals, Osaka, Japan) in water and (B) 0.1% formic acid in methanol (Wako Pure Chemicals). Samples were separated by gradient elution technique: 0\u0026ndash;7 min 5\u0026ndash;90% B, 7\u0026ndash;10 min 90% B, 10\u0026ndash;10.1 min 90\u0026ndash;5% B, and 10.1\u0026ndash;15 min 5% B. The column oven temperature was maintained at 40 \u0026deg;C. The effluent was subjected to mass spectrometry using an electrospray ionization (ESI) interface operating in the positive-ion mode. The source temperature was set at 400 \u0026deg;C, and the ion spray voltage was set at 4.5 kV. Nitrogen was used as a nebulizer and drying gas. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor mass transitions in positive ion mode: CysS-mBB m/z 312.0\u0026rarr;192.0, CysSS-mBB m/z 344.0\u0026rarr;192.0, GS-mBB m/z 498.0\u0026rarr;435.0, GSS-mBB m/z 530.0\u0026rarr;192.0, GSSS-mBB m/z 562.0\u0026rarr;192.0, mBB-S-mBB m/z 415.3\u0026rarr;193, mBB-SS-mBB m/z 447.3\u0026rarr;192, mBB-SSS-mBB m/z 479.3\u0026rarr;192, Sulfite-mBB m/z 271.0\u0026rarr;192.0, Thiosulfate-mBB m/z 303.0\u0026rarr;192.0.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u003c/strong\u003e All statistical analyses were performed using Microsoft Excel 2023 for Window 10 (Microsoft, Redmond, WA, USA) with the Analysis ToolPak add-in software. Differences between two groups were analyzed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data included in this study are available in Supplementary Figures containing detailed statistical analyses. The data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Sciences, Sports, and Technology, Japan to H.K. (23K06016), and by Smoking Research Foundation to H.K. NCNP biobank is partly supported by a grant from Japan Agency for Medical Research and Development (AMED), GAPFREE4 (21ak0101151h0002) and by an Intramural Research Grant (6-1 and 6-7) for Neurological and Psychiatric Disorders of NCNP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.K. and H.K. performed experiments and data analysis, and H.K. supervised the study. Y.K., K.H., Y.O., T.T., C.F., Y.T., and H.K. wrote the manuscript with consultation from all authors. K.H., Y.O., T.T., C.F., and Y.T. interviewed participants and collected cerebrospinal fluid and plasma samples. M.T., and D.I. provided suggestions for LC-MS/MS experiments. T.N. suggested the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated during this study are included in this published article and supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary information\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCirino, G., Szabo, C., Papapetropoulos, A. Physiological roles of hydrogen sulfide in mammalian cells, tissues, and organs. \u003cem\u003ePhysiol Rev.\u003c/em\u003e\u003cstrong\u003e103\u003c/strong\u003e, 31-276. (2023).\u003c/li\u003e\n \u003cli\u003eKumar, R., Banerjee, R. 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Chromatography B.\u003c/em\u003e\u003cstrong\u003e1029-1030\u003c/strong\u003e, 213-221 (2016).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7804547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7804547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) and polysulfides produced by enzymes regulate neuronal transmission and protect neurons against oxidative stress. Abnormalities in their levels have been implicated in the pathophysiology of schizophrenia (SZ), Alzheimer\u0026rsquo;s disease (AD), and Parkinson\u0026rsquo;s disease (PD). However, the levels of these molecules in the cerebrospinal fluid (CSF) and plasma (PLA) obtained from the same individuals with or without smoking habits have yet to be comprehensively studied. Here, we showed that the levels of neuroprotective H\u003csub\u003e2\u003c/sub\u003eS and polysulfides in the CSF were significantly decreased in patients with AD and SZ compared with those in control individuals, suggesting that neuronal activity is not well regulated and that neurons are inadequately protected in both diseases. In contrast, in PD, the levels of these molecules increase specifically in the PLA to unfavorable levels, suggesting peripheral abnormalities, including inflammation. The increased levels of these molecules in PD were restored by smoking habits to the levels in control non-smokers, suggesting that smoking may be linked to a lower risk of developing PD. H\u003csub\u003e2\u003c/sub\u003eS and polysulfides play important roles in the pathophysiology of psychiatric and neurodegenerative diseases, and therefore, are potential therapeutic targets.\u003c/p\u003e","manuscriptTitle":"Hydrogen sulfide and polysulfide levels in cerebrospinal fluid and plasma of patients with schizophrenia, Alzheimer’s disease, Parkinson’s disease, and in relation to cigarette smoking","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 06:29:49","doi":"10.21203/rs.3.rs-7804547/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a10a46b5-a540-499e-996b-12e7d26a10e1","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56878468,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Schizophrenia"},{"id":56878469,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Parkinson's disease"}],"tags":[],"updatedAt":"2025-12-03T10:55:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-29 06:29:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7804547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7804547","identity":"rs-7804547","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}