Methamphetamine (MA) use has created significant public health problems worldwide. It is a psychostimulant that users are prone to abuse because it can be easily synthesized using drugs such as ephedrine and pseudo-ephedrine, which are the raw materials for over-the-counter cold medicines [1]. Acute MA exposure excites the sympathetic nervous system and induces euphoria, enhanced energy, alertness, and libido [2,3]. In contrast, chronic MA exposure can not only induce personality changes such as delusions, violence, and psychotic behavior, but also pulmonary hypertension, stroke, myocardial infarction, and neurocognitive impairments [4,5]. Chronic MA abuse also disrupts neural plasticity and causes neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) [6,7]. Therefore, it is important to identify biomarkers that can be used in the diagnosis and treatment of diseases/impairments caused by MA.

α-synuclein (α-Syn) is a presynaptic protein that is enriched in dopaminergic neurons in the brain and participates in exocytosis, vesicle trafficking, neurotransmitter packing, and regulation of dopamine release [8,9]. Overexpression or aggregation of α-Syn leads to the formation of Lewy bodies, which are intracellular inclusions implicated in the development or progression of PD [10]. Recently, several studies have demonstrated that chronic MA use leads to an increase in the α-Syn level in the brain of MA users and animal models [11-13]. MA not only induces α-Syn accumulation in neurons by dysregulating the ubiquitin-proteasome system and molecular chaperone autophagy but also promotes α-Syn phosphorylation, contributing to neurotoxicity and PD-like pathological changes [14-16]. Beyond its well-established role in PD, α-Syn dysregulation has also been implicated in psychiatric disorders. Elevated serum levels of α-Syn have been reported in patients with major depression disorder, suggesting a potential link between α-Syn and depression [17]. Furthermore, α-Syn accumulation in serotonergic neurons has been associated with mood and emotional disturbances, mirroring neuropsychiatric symptoms observed in PD [18]. These findings suggest that α-Syn dysregulation may contribute to the pathophysiology of certain psychiatric conditions, further emphasizing the need to investigate its role in MA-induced neurotoxicity and psychiatric comorbidities.

Brain-derived neurotrophic factor (BDNF), a neurotrophin, plays an important role in synaptic connectivity and regulation of neuronal outgrowth, differentiation, repair, and survival [19,20]. A decrease in BDNF has been reported to affect the pathology of spinal cord injuries and neurodegenerative diseases including AD and PD [21]. Previous studies have reported that BDNF is involved in substance addiction, including to drugs and alcohol [22-24]. According to a meta-analysis by Ornell et al. [25], people actively using substances such as alcohol and cocaine have a lower level of BDNF in the blood compared to those who are not using such substances. Therefore, a change in the blood level of BDNF in patients with drug dependence (including MA) can be a sign of drug addiction or symptoms of neurodegenerative diseases induced by drug addiction.

Neuron-specific enolase (NSE, γ-enolase), which is predominantly found in the cytoplasm of neurons in the central nervous system (CNS), regulates neuronal glycolysis and is involved in cell survival, energy metabolism, and neuroplastic pathways [26,27]. Two prior studies found that the expression level of NSE was altered in the brains of addictive drug users [28,29]. MA exposure to mesencephalic cultures and monkeys induced a decrease in NSE in both the cultures and hippocampi of the monkeys [30,31]. On the contrary, elevated serum NSE levels have been observed in certain neuropsychiatric conditions among adolescents, such as post-traumatic stress disorder (PTSD) and attention-deficit hyperactivity disorder (ADHD). Adolescents with PTSD exhibited significantly higher NSE levels compared to those with major depressive disorder and healthy controls [32]. Similarly, adolescents with internet addiction comorbid with ADHD or anxiety disorders have been reported to show increased plasma NSE concentrations, suggesting underlying neuronal injury in these populations [33]. Conversely, a study that analyzed cerebrospinal fluid (CSF) and serum of patients with schizophrenia did not find significant elevations, indicating that NSE alterations may not serve as a universal marker across all psychiatric disorders [34]. Therefore, further evaluation of serum NSE in patients with MA use disorder is necessary to explore its potential as a biomarker.

Some studies have shown that the neurotoxic effects of MA induce structural and functional impairments in the blood-brain barrier (BBB) [35,36]. The BBB is an important structure that regulates the homeostasis of the brain microenvironment. The BBB protects the CNS by restricting the free movement of substances between the CNS and blood [37]. Therefore, disruption of the BBB caused by MA neurotoxicity may allow pathogenic agents (e.g., inflammatory factors and virus) to enter the CNS and growth factors and cytokines produced in the brain to enter the bloodstream [38]. On the basis of these prior studies, we hypothesized that expression changes of α-Syn, BDNF, and NSE in the brain due to MA abuse would affect their concentrations in the blood. We measured these concentrations in the blood and confirmed that secretion of α-Syn increased with MA abuse. Therefore, it is possible that α-Syn could be used as a biomarker to diagnose MA abuse.

MA abuse not only disrupts neurotransmitter systems, but also causes neurotoxicity in the brain. In addition, neurotoxicity caused by chronic MA use triggers neurocognitive impairments, leading to the onset of neurodegenerative diseases such as AD and PD. Therefore, it would be very useful if there were blood biomarkers that could identify brain impairments in patients with MA use disorder. In the present study, we investigated the expression levels of three proteins in the blood of patients with MA use disorder to evaluate their utility as biomarkers.

Previous studies have reported that substance use and withdrawal often accompany symptoms of depression [41-43]. According to a self-report, MA users experience psychological symptoms such as anxiety, depression, and suicidal thoughts [41,44]. A previous study found that depression was the most common psychiatric symptom of amphetamine users in Australia [45]. More recently, in a study of subjects experiencing early MA withdrawal (abstinence for 1-7 days), depression was significantly correlated with anxiety [46]. This result demonstrates that MA use induces both depression and anxiety. We similarly found that depression and anxiety symptoms in the MA group (abstinence within 2 weeks) were five times higher than in the control group. Our findings are meaningful because depression and anxiety were compared between the control and MA groups, unlike other research teams that only studied MA-dependent patients [46,47]. Our results also suggest that MA users continue to suffer from depression and anxiety even during initial MA withdrawal. In addition, we observed a significantly lower rate of alcohol use in the MA group compared to the control group. This result may be related to lifestyle changes or behavioral adaptations associated with chronic MA use. MA users may avoid alcohol consumption due to interactions or undesirable combined effects with MA, reflecting a distinct substance-use pattern compared to the general population. However, this discrepancy could also reflect underreporting or variations in the reliability of self-reported alcohol use among individuals with substance abuse disorders. Future studies are warranted to further investigate the impact of concurrent alcohol use on biomarker levels in patients with MA use disorder.

In this study, the α-Syn level was significantly increased in the blood of patients with MA use disorder. Dopaminergic neurons are the main source of α-Syn, which plays a pivotal role in the development or progression of PD through the formation of intracellular inclusions [8,10]. Previous studies have reported that MA treatment increased α-Syn expression within dopaminergic neurons of the substantia nigra in mice [48] and in dopaminergic SK-N-SH cells [49]. Our group also previously observed upregulated expression of α-Syn mRNA in chronically MA-treated monkeys [31]. In contrast, other studies showed that MA led not only to the upregulation and aggregation of α-Syn in many regions of the brain including the hippocampus and olfactory bulb in mice [50], but also to the formation of cytoplasmic multilamellar bodies in PC12 cells [51]. Furthermore, recent findings suggest that MA-induced oxidative stress and neuroinflammation contribute to α-Syn post-translational modifications, including phosphorylation and aggregation, which exacerbate synaptic dysfunction and neurotoxicity [14-16]. Post-mortem studies of chronic MA abusers have similarly revealed increased α-Syn accumulation in the substantia nigra, aligning with these experimental findings [9,13,52]. MA is also known to disrupt the BBB, facilitating the transfer of α-Syn from the brain into the bloodstream [53-56]. This supports the hypothesis that chronic MA exposure not only enhances α-Syn expression in the brain but also contributes to its accumulation in the peripheral circulation, potentially serving as an early indicator of neurodegenerative processes. Taken together, these findings suggest that chronic MA use leads to increased α-Syn levels in both the brain and blood, providing indirect evidence of its role in PD risk. These changes persist even into the withdrawal period, suggesting long-lasting neurobiological consequences.

BDNF participates in neurogenesis and neuroprotection [57]. In addition, BDNF regulates both short- and long-lasting synaptic interactions that play a role in cognition and memory [58]. In the present study, the expression level of serum BDNF did not differ between the MA and normal control groups. This result is consistent with our previous study, in which chronic exposure to MA did not affect BDNF mRNA expression in the hippocampi of monkeys [31]. Although there are species and tissue differences, our results indicate that MA did not significantly affect BDNF expression in either primates or humans. In contrast, other studies that analyzed blood BDNF levels in patients with MA use disorder during MA withdrawal found that patients had higher BDNF concentrations than did healthy controls [59,60]. Previous reports have suggested that BDNF expression increases following CNS impairment, possibly as a compensatory mechanism [61,62]. Based on these findings, Kim et al. [59] proposed that neurotoxicity due to high-dose MA exposure might induce a BDNF increase similar to that observed in CNS-impaired patients during the addiction recovery process. In addition, an in vitro study reported that MA treatment stimulated BDNF expression in primary neurons [63]. Interestingly, in our study, BDNF concentration showed a positive correlation trend with the duration of MA use, although it was not statistically significant. However, when comparing BDNF levels between short-term (≤5 years) and long-term (>5 years) MA use groups, we found that patients with long-term MA use had significantly higher serum BDNF levels than those with short-term MA use (p=0.0178). This suggests that prolonged MA exposure may upregulate BDNF expression, potentially as a compensatory response to MA-induced neurotoxicity. Although the role of BDNF in substance addiction remains controversial, our findings support the idea that BDNF expression is associated with the duration of MA use within patients with MA use disorder. This aligns with previous reports suggesting that chronic drug exposure may trigger neuroplasticity-related changes in BDNF regulation. Further studies are needed to determine whether BDNF upregulation in long-term MA users reflects a neuroprotective adaptation or an alternative underlying mechanism of addiction-related neurobiological changes.

As described previously, NSE is responsible for neuroplastic pathways as well as neuronal glycolysis in the brain, and its level is affected by addictive drug use [28,29]. In a study of Liao et al. [28], the level of NSE mRNA decreased in immortalized lymphoblastoid cell lines that originated from subjects with heroin dependence. In our previous study, acute high-dose exposure and chronic exposure of MA to cynomolgus monkeys also led to a decrease in the NSE mRNA level in the hippocampus [31]. These two studies imply that the NSE level may be reduced in the blood of patients with MA use disorder. Unfortunately, in this study, the level of NSE did not change in the blood of patients with MA use disorder. However, there tended to be a negative correlation between the duration of drug administration and the NSE level in patients, although it was not significant. Given that blood samples were collected from patients who had been abstinent from MA for more than 24 hours, NSE concentrations may vary depending on the duration and progression of withdrawal.

In summary, we investigated whether blood proteins associated with neurodegeneration and neural plasticity changed in patients with MA use disorder after MA withdrawal. We identified an increase in the α-Syn level in the blood of patients with MA use disorder, even during MA withdrawal. This result suggests that α-Syn increases in the brain after exposure to MA (because it crosses the BBB). We also found that the BDNF level increases with prolonged exposure to MA, implying it as a compensatory mechanism after brain injury. Nevertheless, this study has some limitations. First, the sample size was relatively small, which may have limited the statistical power to detect subtle differences in protein expression. To mitigate this limitation, we conducted statistical assessments such as Q-Q plots, histograms, and normality tests to evaluate data distribution and ensure the appropriateness of parametric or nonparametric statistical analysis. These methods helped validate our findings despite the sample size constraint. However, future studies with larger cohorts are necessary to confirm our results and improve the generalizability of these findings. Second, variability in withdrawal durations may have influenced biomarker levels. While all patients with MA use disorder had undergone withdrawal for 1 to 2 weeks prior to participation, differences in protein expression may still exist depending on individual variations in withdrawal severity and progression. Future studies should account for the effects of withdrawal duration and symptom severity to better understand MA-induced neurobiological changes. Third, despite matching the control and MA groups for age, sex, and smoking status, significant differences in socioeconomic status and education level remained. These factors may act as confounders, as socioeconomic disparities influence health and neurobiological functioning. Fourth, this study relied on peripheral blood biomarkers to infer neurotoxicity, which has inherent limitations. Although α-Syn, BDNF, and NSE are linked to neurodegenerative changes, their direct correlation with CNS pathology remains uncertain. Peripheral biomarker levels can be affected by systemic factors such as metabolic and inflammatory responses, potentially confounding interpretations of MA-related neurotoxicity. Future studies should incorporate neuroimaging or CSF analysis for a more comprehensive assessment of CNS changes in MA use disorder. Despite these limitations, the observed increase in peripheral α-Syn levels in patients with MA use disorder suggests its potential as a biomarker for MA-induced neurotoxicity and neurodegenerative processes. Further research is warranted to validate α-Syn as a biomarker for MA-related brain dysfunction and explore its role in assessing long-term neurobiological consequences of MA abuse.