Methamphetamine (METH) is a highly addictive psychostimulant that causes long-lasting effects in the brain and increases the risk of developing neurodegenerative diseases. The cellular and molecular effects of METH in the brain are functionally linked to alterations in glutamate levels. Despite the well-documented effects of METH on glutamate neurotransmission, the underlying mechanism by which METH alters glutamate levels is not clearly understood. In this study, we report an essential role of proline biosynthesis in maintaining METH-induced glutamate homeostasis. We observed that acute METH exposure resulted in the induction of proline biosynthetic enzymes in both undifferentiated and differentiated neuronal cells. Proline level was also increased in these cells after METH exposure. Surprisingly, METH treatment did not increase glutamate levels nor caused neuronal excitotoxicity. However, METH exposure resulted in a significant upregulation of pyrroline-5-carboxylate synthase (P5CS), the key enzyme that catalyzes synthesis of proline from glutamate. Interestingly, depletion of P5CS by CRISPR/Cas9 resulted in a significant increase in glutamate levels upon METH exposure. METH exposure also increased glutamate levels in P5CS-deficient proline-auxotropic cells. Conversely, restoration of P5CS expression in P5CS-deficient cells abrogated the effect of METH on glutamate levels. Consistent with these findings, P5CS expression was significantly enhanced in the cortical brain region of mice administered with METH and in the slices of cortical brain tissues treated with METH. Collectively, these results uncover a key role of P5CS for the molecular effects of METH and highlight that excess glutamate can be sequestered for proline biosynthesis as a protective mechanism to maintain glutamate homeostasis during drug exposure.
Methamphetamine (METH) is a powerful and highly addictive psychostimulant1. It belongs to a larger group of drugs called Amphetamine-Type Stimulants (ATS)1,2,3. Use of ATS generate a sense of euphoria, increase energy and concentration as well as decrease appetite, induce weight loss, leading to a variety of emotional, cognitive, and physical effects4,5. Among the ATS, METH has the greatest potential for abuse because of easy availability, low cost, and longer duration of action1,2,3. Therefore, METH use and abuse continues to be a significant public health problem in the United States and all over the world1,2,3.
METH is a lipophilic compound that crosses the blood–brain barrier easily to cause long lasting effects in the brain4,5. Since METH’s chemical structure is similar to monoamines, it targets both dopaminergic (DA) and serotonergic (5HT) neurons in the brain. METH enters these neurons by binding to the membrane-bound DA transporter (DAT) and/or 5HT transporter3. There is also evidence that METH can cross neuronal membranes by passive diffusion mechanism6. Once inside the neuron, METH can alter the function of the vesicular monoamine transporter 2 (VMAT-2) and affect cytoplasmic monoamine concentrations and DA release6. METH administration is also known to increase DAT internalization and affect 5HT transporter functionality6,7,8. In addition, METH prevents degradation of neurotransmitters, reduces neurotransmitter reuptake, and causes an efflux of DAT and 5HT transporters6,7,8. These biochemical and cellular effects result in an increased neurotransmitter availability in the synapse6,7,8 and cause continuous stimulation of neurons to manifest the euphoric effects among METH users9,10,11.
In addition to its effects on DA and 5HT neurons, METH has been shown to affect glutamate neurotransmission12,13. There is evidence that METH’s effect on glutamate neurotransmission is mediated by increasing levels of extracellular glutamate13,14. However, the mechanism of glutamate homeostasis during METH exposure has not been clearly delineated. Glutamate is the principal excitatory neurotransmitter and is ubiquitously distributed in the brain15,16,17. Due to its critical role in neuronal plasticity, glutamate is involved in a number of critical brain functions15,16,17 including learning and memory18. It is important to note that glutamate is produced in the nerve terminals predominantly from two sources: (1) the tricarboxylic acid (TCA) cycle, and (2) glutamine produced by the glial cells19,20. Glutamate produced in the nerve terminals is packaged into vesicles by the vesicular glutamate transporters (vGLUT) and is released to the extracellular space upon stimulus of an action potential20. After release, the glial cells clear glutamate from the synapse via the glutamate transporters, GLAST and GLT-1, to maintain sub-neurotoxic levels of extracellular glutamate21,22,23. Maintaining glutamate homeostasis is critical for proper neuronal function since excessive glutamate has been implicated in neuronal excitoxicity24.
The link between glutamate metabolism and carbohydrate/amino acid metabolism has been well established. Specifically, glutamate is channeled to the TCA cycle—a common pathway in carbohydrate and amino acid metabolism25. However, the metabolic link between glutamate and proline26,27 remains largely understudied. Proline, unlike other amino acids, has its α-amino group within a pyrrolidine ring, and thus it is the sole proteinogenic secondary (imino) amino acid27. Proline is also unique since it has its own set of metabolic enzymes28. It is known that glutamate can be converted to proline through Δ1-pyrroline-5-carboxylate (P5C) by the enzymatic activity of P5C synthase (P5CS) and P5C reductase (PYCR)26,29 (Fig. 1A). Conversely, proline can also be converted to glutamate through the catabolic pathway catalyzed by proline oxidase/dehydrogenase (POX/ PRODH) and P5C dehydrogenase (P5CDH)25,26,29. Given this important metabolic link, proline metabolism has been reported to play key roles in normal brain function and various neurological disorders.
Proline is abundantly found in the central nervous system (CNS)30. The presence of high affinity proline transporters has also been reported in a subset of glutamatergic neurons in rodent brain31. In animal models, elevated proline levels affect glutamatergic transmission32, depolarize neurons33,34, increase synaptic activity35, alter cognitive tasks, sensorimotor gating, and locomotor activity36,37. In humans, hyperprolinemia—a condition associated with abnormally elevated levels of proline has been linked to epilepsy, seizures and impaired cognitive function38. Hyperprolinemia is also linked to schizoaffective disorders39 and schizophrenia40,41. Even though these studies and others demonstrate key roles of proline in brain function and neurological disorders, the role of proline metabolism during drug exposure remains largely unknown.
In this study, we report a functional link between proline and glutamate metabolism during acute METH exposure. First, we utilized the SH-SY5Y neuronal cell model, that has been widely used to study neuronal biology42,43. SH-SY5Y cells are positive for tyrosine hydroxylase (TH) and dopamine-β-hydroxylase characteristic of catecholaminergic neurons that are known to be affected by METH44. In this model, we tested the functional contribution of proline metabolism in METH-exposure mediated alterations in glutamate levels. Interestingly, acute treatment with METH markedly induced the enzymes of the proline biosynthetic pathway. Specifically, a marked induction in the expression of P5CS and PYCRs was observed in METH treated neuronal cells. This induction was accompanied by a concomitant increase in intracellular proline levels. Surprisingly, METH exposure neither increased extracellular glutamate levels or caused neuronal excitotoxicity. However, blocking the proline synthetic arm by knocking-out P5CS increased both intracellular and extracellular glutamate levels in METH treated cells. Furthermore, treatment of proline auxotrophic-Chinese hamster ovary (CHO-K1) cells with METH significantly enhanced extracellular glutamate levels. These results suggested a functional link between biosynthesis of proline and glutamate during METH exposure. Finally, levels of proline biosynthetic enzymes, specifically, P5CS and PYCR2 were also significantly induced in the cortex of animals administered with METH and in the slices of cortical brain tissues exposed to METH. Collectively, our studies demonstrate activation of proline biosynthesis to sequester increased glutamate during acute METH exposure and imply an essential role of proline metabolism in limiting neuronal glutamate excitotoxicity.
Acute METH exposure activates the proline synthetic pathway
METH exposure affects glutamate neurotransmission primarily by altering glutamate levels12,13. While, glutamine serves as the major source of glutamate21,22,23, proline metabolism is also closely linked to glutamate25 (Fig. 1A). However, the functional link between proline and glutamate during drug exposure induced glutamate neurotransmission is poorly understood. Therefore, we examined the effects of METH exposure on proline metabolism using the SH-SY5Y neuronal cell model. First, we measured the levels of enzymes of the proline catabolic arm- POX and P5CDH- which sequentially convert proline to P5C and then to glutamate (Fig. 1A). We treated SH-SY5Y cells with METH in a dose dependent manner using concentrations of 1 mM and lower. Although higher concentrations of METH have been used in published studies45,46, we chose concentrations below 1 mM since higher levels of the drug are rarely achieved in METH associated disorders47,48. Lysates of METH-treated and control cells were analyzed by immunoblot to measure the levels of POX and P5CDH. These analysis revealed that SH-SY5Y cells endogenously express all the key metabolic enzymes of proline (Fig. 1B,D). Interestingly, METH exposure minimally altered the expression of POX and P5CDH when compared to the untreated controls (Fig. 1B–D). Even at 1 mM concentration of METH, expression of these two enzymes were not significantly altered (Fig. 1B–D), suggesting that acute METH exposure minimally alters the proline catabolic arm that converts proline to glutamate (Fig. 1A).
We next measured the expression of enzymes of the proline biosynthetic arm (Fig. 1A), P5C synthase (P5CS) and P5C reductase (PYCR) that convert glutamate to proline by sequential catalysis26. Notably, the expression of P5CS was significantly increased in cells exposed to METH (Fig. 1E,F). Densitometry analysis illustrated a dose-dependent increase in P5CS expression in cells treated with METH relative to untreated cells (Fig. 1F). Then, we measured the levels of PYCRs- PYCR1, and PYCR2, which are involved in biosynthesis of proline (Fig. 1A)27,29,49. As presented in Fig. 1E,G,H, expression of the PYCR1 and PYCR2 was significantly increased in the METH treated cells. While the expression of PYCR1 was increased ~ 1.7-fold, the levels of PYCR2 was ~ sixfold higher in cells treated with 1 mM METH compared to the untreated control cells (Fig. 1E,G,H). Collectively, these results demonstrated that acute METH exposure activates the proline synthetic arm without altering the catabolic arm.
METH exposure upregulates key proline metabolic enzymes in differentiated neuronal cells and cortical brain slices
Our results in Fig. 1 showed upregulation of proline synthetic enzymes by METH exposure in undifferentiated SH-SY5Y cells. Even though the SH-SY5Y neuroblastoma cells show some characteristics of neurons, we have previously shown that these cells acquire a neuronal phenotype by differentiation utilizing ATRA50. Therefore, we used differentiated SH-SY5Y cells and treated them with METH in a dose dependent manner. 24 h post treatment cellular extracts were prepared to measure the expression of P5CS and PYCR2, as representatives of proline synthetic enzymes. Immunoblot analysis showed that METH treatment resulted in a significant upregulation of both P5CS and PYCR2 when compared to untreated controls (Fig. 2A–C). These observations are similar to the results obtained with the undifferentiated cells (Fig. 1). To further probe the physiological relevance of these cell-based observations, we carried out METH exposure experiments using slices of cortical regions of rodent brain. Given the technical challenges associated with brain slice experiments, 200 µM METH was selected based on the significant upregulation of both P5CS and PYCR2 in neuronal cells at this concentration (Figs. 1E–H, 2A–C). Brain slices were incubated in METH or vehicle (saline) for a 6-h period. Then cortical enriched tissue was isolated and immunoblot analysis was carried out of the tissue lysates. Data from these studies illustrated that P5CS and PYCR2 levels were significantly upregulated in the cortical brain slices upon METH exposure (Fig. 2D–F). Taken together, results in Figs. 1 and 2A–F establish that acute METH exposure upregulates the proline metabolic enzymes in neuronal cells and cortical brain slices.
Next, we determined whether activation of proline synthetic arm is specific to METH exposure or a general mechanism utilized by other ALS. To test this, we first treated undifferentiated SHSY-5Y cells with increasing concentration of Amphetamine (AMPH). Immunoblot analysis of cellular lysates showed a minimal but non-significant increase in P5CS and PYCR2 protein expression by AMPH exposure when compared to untreated controls (Fig. 2G–I). Interestingly, AMPH exposure of differentiated SH-SY5Y cells illustrated a modest increase in the expression of P5CS at 1000 µM METH. However, PYCR2 expression was minimally changed in the differentiated cells exposed to increasing concentrations of AMPH (Fig. 2J–L). Finally, exposure of brain slices to AMPH (200 µM) showed a lack of significant increase in the expression of both P5CS and PYCR2 (Fig. 2L–O). Collectively, results described in Fig. 2 strongly suggested that acute METH exposure activates the proline catabolic arm, whereas AMPH exposure lacks such activation.
Acute METH exposure enhances proline levels but does not increase glutamate levels
Results in Figs. 1 and 2 demonstrated that METH exposure upregulated the expression of P5CS and PYCR2. Since these enzymes catalyze the synthesis of proline from glutamate, we measured intracellular proline levels in METH treated SH-SY5Y cells. We chose 200, 500 and 1000 μM of METH for this experiment since both P5CS and PYCR2 were significantly upregulated at these drug concentrations (Figs. 1, 2). To quantify proline levels, we utilized the acid-ninhydrin assay that specifically detects proline51,52. Results from this assay illustrate that the levels of intracellular proline were significantly elevated in METH treated cells when compared to the untreated control cells (Fig. 3A). Specifically, treatment of cells with 200 μM METH resulted in a ~ 100 μM increase in proline levels relative to the untreated cells. Similarly, treatment with 500 μM METH further increased the proline levels up to ~ 130 μM and 1000 μM METH enhanced proline levels up to ~ 155 μM. These results indicate that acute METH exposure significantly enhances proline biosynthesis in accordance with the induction of proline synthetic enzymes.
A number of studies have shown that METH exposure alters glutamate levels53,54,55. Therefore, we tested whether the increased proline levels were due to higher glutamate in METH-treated cells. We measured the levels of both intracellular and extracellular glutamate in SH-SY5Y cells. We treated these cells with 50–1000 µM METH for 24 h. Following that, we measured glutamate levels in the cellular lysates and supernatants by a colorimetric assay. As shown in Fig. 3B,C, treatment of SH-SY5Y cells with METH at concentrations of 0–1000 µM did not significantly increase either intracellular (Fig. 3B) or extracellular (Fig. 3C) glutamate levels. Surprisingly, there was a slight decrease in both intracellular and extracellular glutamate levels (~ 20–30%) in cells treated with METH at the concentrations 200–1000 µM. Collectively these results suggest that acute METH treatment at concentrations ≤ 1 mM minimally affects intracellular and extracellular glutamate levels.
A lack of increased glutamate in METH treated cells could be due to reduced glutamate synthesis from glutamine-the major source of glutamate in neurons (19,20). To test this hypothesis, we measured the levels of glutaminase (GLS) enzyme that converts glutamine to glutamate in neurons (22–24). Western blot analysis revealed that METH treatment did not reduce GLS levels in SH-SY5Y cells, (Fig. 3D,E). Interestingly, a modest increase in GLS expression was observed in METH treated cells (Fig. 2D,E). These observations suggested that glutamate synthesis is not reduced in METH treated cells. Thus, a lack of increased glutamate during acute METH exposure was most likely not due to reduced synthesis from glutamine.
Extracellular glutamate levels are also dependent on packaging of intracellular glutamate by the vesicular glutamate transporters: vGLUT1, vGLUT2, and vGLUT3 (22–24). Among these transporters, the functional characteristics of vGLUT1 and vGLUT2 for glutamate transport is similar56. Interestingly, vGLUT3 has been reported to be found in cholinergic neurons but not in glutamatergic cell populations57. Therefore, we measured the levels of vGLUT1 as the selected marker of glutamate release in METH treated cells. Surprisingly, western blot analysis revealed a dose-dependent decrease in vGLUT1 expression with increasing concentration of METH (Fig. 3D,F). The reduction in vGLUT1 expression suggested that the packaging of glutamate was most likely reduced in METH treated cells. Interestingly, the reduction in vGLUT1 expression is correlated with the decrease in extracellular glutamate levels in cells treated with METH at concentrations of 200–1000 µM (Fig. 3C). Finally, we also measured neuronal cytotoxicity by LDH assay in the supernatants of METH-treated cells. METH treatment did not cause any cytotoxicity even at 1 mM concentration when compared to untreated control cells (Fig. 3G). Collectively, these results demonstrate that acute METH exposure at concentrations below 1 mM significantly enhances proline levels but minimally increases extracellular glutamate and lack the ability to cause neuronal cytotoxicity.
P5CS is the key enzyme that regulates glutamate levels during acute METH exposure
Our results in Fig. 3A demonstrated that acute METH exposure activates proline biosynthesis. However, a lack of alterations in glutamate levels in these cells (Fig. 3B,C), led us to hypothesize that activation of proline biosynthesis may aid in regulating glutamate levels during acute METH exposure. To test this, we created SH-SY5Y cells that are deficient in proline biosynthesis from glutamate. We specifically depleted the P5CS enzyme since synthesis of proline from glutamate is regulated by P5CS (Fig. 1A)26 and METH significantly increased P5CS levels (Figs. 1, 2). Even though PYCR2 was also upregulated by METH, blocking the reductase step required depletion of both PYCR1 and PYCR2 due to the functional redundancy of these two enzymes. Therefore, we generated P5CS knockout SH-SY5Y cells (P5CS-KO) using CRISPR/Cas9 genome-editing approach58. We employed a lentiviral vector-based expression of nuclease-active Cas9 and P5CS gRNAs and selected single clones of P5CS-KO. Western blot analysis confirmed lack of P5CS expression in the P5CS-KO cells when compared to the wild type cells (Fig. 4A). We then treated the P5CS-KO cells with METH in a dose-dependent manner for 24 h. Measurement of glutamate levels showed a significant increase in intracellular glutamate levels in the P5CS-KO cells upon METH treatment (Fig. 4B). For instance, METH exposure at 50 µM resulted in ~ 1.8-fold increase in intracellular glutamate levels in P5CS-KO cells compared to the untreated control. This increase was further enhanced to ~ 2.6-fold with 1 mM METH treatment P5CS-KO cells (Fig. 4B). Interestingly, acute METH exposure also significantly elevated extracellular glutamate levels in the P5CS-KO cells (Fig. 4C). These results are in contrast to the minimal effect of METH exposure on glutamate levels in the wild type cells that express P5CS (Fig. 3B,C).
To further solidify the role of P5CS in regulating glutamate levels, we probed the expression of PYCR2 in the P5CS-KO cells, since METH treatment increased PYCR2 expression in wild type cells (Figs. 1, 2). Results presented in Fig. 4D,E clearly show that METH exposure minimally alters the expression of PYCR2 in the P5CS-KO cells. Moreover, in P5CS-KO cells treatment of METH minimally affected GLS expression (Fig. 4F,G), suggesting that depletion of P5CS does not affect glutamate synthesis from glutamine. Interestingly, a marginal increase in vGLUT1 expression was observed in the P5CS-KO cells upon METH treatment above 500 µM (Fig. 4H). This increase is consistent with the increased glutamate levels observed in the P5CS-KO cells after METH exposure. Collectively, these results establish that depletion of P5CS causes accumulation of glutamate and strongly suggest the involvement of proline biosynthetic arm in maintaining METH-induced glutamate homeostasis.
METH exposure increases glutamate levels in proline-auxotrophic Chinese hamster ovary (CHO) cell line
Our results demonstrated that P5CS expression is associated with alterations in glutamate levels during acute METH exposure (Fig. 4). To further probe a functional link between proline synthetic arm and glutamate, we exploited the proline auxotrophic nature of Chinese Hamster Ovary (CHO) cells. CHO cells lack the proline biosynthetic arm that is needed to catalyze proline from glutamate59,60. Specifically, these cells do not express P5CS and PYCR2 as measured by western blot analysis (Fig. 5A). Therefore, to further strengthen the role of P5CS during glutamate homeostasis, CHO cells were treated with increasing doses of METH for 24 h. Then extracellular glutamate levels were measured in the culture supernatants of treated and untreated cells. As shown in Fig. 5C, a dose-dependent increase in glutamate levels was detected in the presence of increasing concentrations of METH.
Next, we tested whether the higher levels of glutamate in METH-treated CHO cells is a consequence of lack of proline synthesis from glutamate. We exogenously expressed P5CS in the CHO cells by transfecting a P5CS expression construct. Post-transfection, western blot analysis of the cellular lysates of these cells confirmed the expression of P5CS compared to the lack of P5CS expression in wild type cells (Fig. 5B). Then, the P5CS-expressing CHO cells were exposed to increasing concentrations of METH for 24 h and extracellular glutamate levels were measured in the supernatants. Results from these assays show that P5CS expression significantly abrogates the increase in extracellular glutamate upon METH exposure as compared to the P5CS-lacking wild type cells (Fig. 5C). Taken together, these observations provide strong evidence that P5CS regulates glutamate levels during acute METH exposure.
Acute METH exposure activates proline biosynthetic pathway in the cortical regions of mice brain
Our studies using neuronal cell model and proline auxotrophic cells (Figs. 1, 2, 3, 4, 5) demonstrated a key role of proline biosynthetic pathway in regulating glutamate levels during acute METH exposure. To determine the in vivo significance of these observations, we investigated the effects of METH exposure on the proline synthetic pathway in a rodent model. Mice were administered with either METH (2 mg/kg IP) or saline under acute conditions for 24 h. Then, the cortical regions of the brain were isolated and total protein from these brain tissues were extracted and subjected to western blot analyses. We focused on the cortical regions, since glutamatergic neuronal projections originating in the prefrontal cortex (PFC) extend to the striatum, nucleus accumbens (NAc), ventral tegmental area (VTA) and substantia nigra of the midbrain61,62 and METH has been shown to affect glutamate in the PFC of mice brain63. Data in Fig. 6A show that P5CS is constitutively expressed in the cortical region of mouse brain. Upon acute METH treatment of these mice, a significant increase in P5CS levels was detected when compared to the control animals, (Fig. 6A,B), suggesting upregulation of P5CS during METH exposure. Furthermore, protein levels of the PYCR2 were also increased in the cortical tissues of METH administered animals (Fig. 6C,D). However, the level of increase in PYCR2 was lower when compared to the significant increase in P5CS levels. These data are in accordance with the results obtained with the neuronal cell model (Figs. 1, 2). Collectively, these observations support the activation of the proline synthetic arm in the cortical region of mice brain during acute METH exposure.
Glutamate is the key excitatory neurotransmitter in the brain15,16,17. During glutamatergic neurotransmission, glutamate is packaged in presynaptic neurons and released into the synapse15,16,17. The released glutamate binds to ionotropic and metabotropic receptors on postsynaptic neurons to propagate the incoming signals15,16,17. Subsequently, high-affinity transporters present on the glial cells and neurons rapidly sequester the glutamate to facilitate a continuous cycle of neurotransmitter activity64. Given the central role of glutamate in neurotransmission, it is involved in most aspects of normal brain function including cognition, memory, and learning18. Glutamate also plays a critical role in drug reward, reinforcement, and relapse65,66,67,68. Importantly, recent studies suggest that in individuals with substance use disorder, glutamate is associated with synaptic plasticity65,66,67,68. Specifically, METH has been reported to affect glutamate neurotransmission by altering glutamate levels14. However, the mechanism by which METH exposure affects glutamate homeostasis is poorly understood.
METH has been shown to alter the extracellular concentrations of both glutamate and dopamine in the brain13. For example, chronic treatment with high doses of METH has been reported to increase glutamate efflux in the striatum14. Depletion of striatal dopamine levels with low dose METH has been shown to enhance glutamate release69. Similarly, a low dose of METH has been demonstrated to increase extracellular dopamine but not glutamate in PFC and striatum63. Additionally, repeated METH administrations were shown to enhance cortical glutamate efflux63. Interestingly, a single injection of a high dose of METH (30 mg/kg, i.p.), was reported to cause glutamate depletion70. These observations suggest that alterations in the levels of glutamate are dependent on the duration and dose of METH exposure. Accordingly, our results with a neuronal cell model demonstrated that acute METH exposure at concentrations below 1 mM minimally altered extracellular glutamate levels. It is noteworthy that majority of the published studies examined the effects of long-term exposure and relatively high doses of METH on glutamate47,71. Therefore, based on our results we speculate that a single dose of METH below 1 mM concentrations causes minimal changes in glutamate homeostasis.
Neuronal glutamate levels are regulated by a multi-prong mechanism that involve glutamate biosynthesis from glutamine and synaptic vesicle loading of intracellular glutamate. Glutamate synthesis is dependent on the glutamine–glutamate cycle between astrocytes and neurons72. In this metabolic cycle, GLS is primarily responsible for synthesizing glutamate from glutamine to continuously replenish the neurotransmitter pool (22–24). While, GLS regulates glutamate biosynthesis from glutamine (22–24), vGLUTs mediate the transport of glutamate into synaptic vesicles73,74 and vGLUT expression determines the amount of glutamate packaged into vesicles73,74. Specifically, in glutamatergic neurons the functionally similar vGLUT1 and vGLUT2 are responsible for glutamate transport56. Interestingly, our results demonstrated that expression of GLS remains mostly unaffected in METH treated cells (Fig. 3). Moreover, with acute METH treatment, a minimal to marginal reduction in vGLUT-1 expression was detected (Fig. 3). These observations strongly suggest that acute METH exposure minimally alters ongoing glutamate biosynthesis and vesicular packaging, consistent with the minimal effect of METH on glutamate levels under acute exposure conditions.
The glutamine–glutamate biosynthetic cycle is not stoichiometric, but is an open biochemical pathway wherein glutamate interfaces with several other metabolic pathways including carbohydrate and amino acid metabolism through the TCA cycle70. In this context, our results describing activation of proline synthetic arm by METH exposure highlights the novel role of proline biosynthesis during METH exposure. Glutamate can be channeled for proline synthesis through P5C. These biochemical reactions are sequentially catalyzed by P5CS and PYCR (Fig. 1A). Remarkably, our data showed that acute METH treatment resulted in a marked enhancement in both P5CS and PYCR2 expression (Figs. 1, 2). Concomitant increase in intracellular proline levels (Fig. 3A) indicated that induction of these two enzymes are most likely responsible for synthesizing proline from glutamate. Moreover, this increase was observed only with METH and not AMPH indicating the effect to be specifically mediated by METH. Previously, a genome-wide study, analyzed METH-induced mRNA expression in rodent brain to reveal the potential regulatory consequences in response to METH75. Pathways upregulated by METH included proline metabolism especially the proline synthetic enzyme P5CS (encoded by ALDH18A1) and PYCRs75. Similarly, a metabolomic study of brain tissues from METH affected animals uncovered neurochemical signatures related to the metabolism of amino acids including glutamate and proline76. Similar to METH, morphine is also known to affect glutamatergic neurotransmission77,78. In a proteomics analysis, PYCR2 was identified as one of the proteins that was induced by morphine in mouse hippocampal postsynaptic density-associated proteins (HPSD)79. These studies indicate an interplay between glutamate and proline metabolism. However, the functional relevance of proline metabolism during METH exposure is unknown. Therefore, our studies showing a functional association between glutamate and proline metabolism during METH exposure are highly significant.
Our results provide strong evidence of glutamate efflux to proline biosynthesis. Specifically, a marked induction in the expression of P5CS was observed during acute METH exposure not only in the SH-SY5Y neuronal cells but also in brain cortical region of mice. Interestingly, disruption in proline biosynthesis by knocking out of P5CS dramatically increased both intracellular and extracellular glutamate with METH treatment. The role of P5CS was further confirmed using a proline auxotrophic cell line (CHO-K1) that lacks the proline biosynthetic machinery. A marked enhancement in glutamate levels was also observed in these cells with METH treatment and this effect was abrogated by overexpression of P5CS. Collectively, these results demonstrated sequestration of neuronal glutamate pool towards proline biosynthesis. A lack of neuronal cell death by acute METH exposure even at 1 mM concentrations supports the hypothesis that sequestration of glutamate to proline is a critical mechanism activated to avert the accumulation of excess glutamate and prevent excitotoxicity.
Sequestration of glutamate to proline may have significant implications in the CNS. For instance, P5C formed during proline synthesis from glutamate can serve as the source for carbon exchange between the TCA and urea cycle28. Proline can also be deposited in collagen, the most abundant protein by weight in the human body80. Approximately, 25% of proline residues make up collagen, therefore, collagen has been suggested to be both a dump as well as a reservoir for proline80. This may be very relevant in the context of the extracellular matrix (ECM) of neurons. For example, excess glutamate can be converted to proline and deposited into the ECM as collagen without dramatically altering extracellular proline levels. High levels of proline have been shown to have excitotoxic properties and studies have shown that addition of proline to hippocampal slices decreased glutamate uptake causing glutamate to accumulate81. It is plausible that once the threshold for proline deposition is reached, especially with chronic METH, it may result in the accumulation of proline, which may also contribute to the glutamate excitotoxicity. Thus, the interplay between glutamate and proline may be an important regulator in METH-induced glutamate excitotoxicity.
Importantly, we envision that increased conversion of glutamate to proline offers a bioenergetic metabolic advantage. The bioenergetic cost of maintaining homeostatic levels of synaptic glutamate is expensive82. Glucose via glycolysis provides the main source of energy for maintaining glutamate homeostasis in the brain. However, for efficient glycolysis a sustained supply of electron acceptor nicotinamide adenine dinucleotide (NAD+) is essential83,84. Proline biosynthesis has been shown to augment glycolysis by affecting the levels of NAD+, especially in cancer metabolism85,86,87. Therefore, our results demonstrating METH-induced proline biosynthesis may confer a metabolic bioenergetic advantage to neurons in response to increased neuronal stimulus by METH. In summary, our results identify a key role of P5CS during METH exposure and highlight that sequestering excess glutamate for proline biosynthesis is a key mechanism to maintain glutamate levels (Fig. 7).
Materials and methods
All the methods used in this study are in accordance with relevant guidelines and regulations.
Methamphetamine hydrochloride (METH), Amphetamine hydrochloride (AMPH), all-trans-retinoic acid (ATRA) were obtained from Sigma-Aldrich Chemicals (USA). The primary antibodies used were as follows: anti-P5CS (cat# NBP1-83324) was purchased from Novus Biologicals (USA), anti-PYCR1 (cat# 13108-1-AP, anti-PYCR2 human (cat# 55060-1-AP, anti-PYCR2 mouse (cat #17146-1-AP, anti-P5CDH (cat# 11604-1-AP), anti-GLS (cat# 12855-1-AP) were purchased from Proteintech (USA), anti-POX IgG was a gift from Dr. James Phang (NCI-Frederick), anti-vGLUT1 (cat# 48-2400) was purchased from Thermo Fisher (USA), and anti-β-actin was obtained from Sigma-Aldrich Chemicals (USA). The secondary antibodies used were goat anti-rabbit or goat anti-mouse purchased from BioRad laboratories (USA).
Cell culture and METH treatment
Human neuroblastoma cells (SH-SY5Y), Chinese hamster ovary (CHO-K1) cells were purchased from American Type Culture Collection (Manassas, VA). The SH-SY5Y cells were maintained in Dulbecco's modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 2 mM Glutamine (Gibco), and 1000 U/mL penicillin, and 100 mg/mL streptomycin (Gibco). CHO-K1 cells were grown and maintained in Ham’s F-12K medium with 10% FBS, 2 mM Glutamine, 1000 U/mL penicillin, and 100 mg/mL streptomycin. SH-SY5Y were differentiated with ATRA as per published methods (48). All cells were maintained at 37 °C and 5% CO2 before and during treatments. The cells were treated acutely for 24 h with METH in a dose dependent manner at physiologically relevant concentrations of 50–1000 μM.
All three P5CS-targeting sgRNA sequences used in this study were from Doench et al.88 and each sgRNA was individually cloned into the TLCV2 plasmid vector (Addgene) by following the recommended protocol with modifications58. Briefly, the TLCV2 plasmid was first digested with BsmB1 by assembling a reaction mixture containing the plasmid DNA, Buffer 3.1 (NEB), and BsmB1 (NEB) and incubating at 55 °C for 30 min, then dephosphorylated by incubating the restriction digestion products with rSAP (NEB) at 37 °C for 30 min, and finally gel-purified. Each pair of oligos corresponding to a sgRNA was phosphorylated and annealed by assembling a reaction mixture containing the oligos, T4 ligation buffer (NEB), and T4 Polynucleotide Kinase (NEB) and incubating at 37 °C for 30 min followed by incubation at 95 °C for 5 min and then ramping down to 25 °C at 5 °C/min. The annealed oligos were diluted 200 fold and then ligated to the BsmB1-digested and dephosphorylated TLCV2 in a ligation mixture containing T4 ligation buffer (NEB) and T4 DNA ligase (NEB) and incubating at room temperature for 1 h. Subsequently, NEB-Stbl competent cells, prepared using the Mix&Go Transformation Kit (Zymo Research, USA) were transformed with an aliquot of the ligation reaction products as per manufacturer-recommended protocol and the bacterial recombinants were confirmed by Sanger sequencing of the isolated plasmids.
To construct the P5CS overexpression plasmid, the P5CS ORF was obtained by RT-PCR (abm, Canada) from SupT1 total RNA using P5CS-specific primers (forward: 5-TTA TAG GAT CCG CCA CCA TGT TGA GTC AAG TTT ACC-3′ and reverse: 5′-TGA CAC TCG AGT CAG TTG GTG TTT CTC TGA G-3′) containing BamH1 and Xho1 restriction enzyme sites. The BamH1 and Xho1 digested P5CS ORF amplicon was ligated to compatible ends of the pcDNA 3.1 plasmid (Addgene). The recombinant plasmids were confirmed by colony PCR and Sanger sequencing.
HEK293T cells (5 × 105) seeded per well in 6-well plates and cultured overnight were transfected with plasmid DNAs using the PEI transfection reagent (Polysciences Inc.). For transfecting cells in each well, a 4:3:1 ratio of lentiviral transfer plasmid (described above), packaging plasmid psPAX2, and pseudotyping envelope plasmid pMD2.G, and the PEI at a DNA:PEI ratio of 1:3 were used. After overnight culturing, the culture medium was removed and the cells were replenished with fresh medium. Forty-eight hours post transfection, the lentivirus-containing culture medium was collected, centrifuged at 500×g for 5 min at room temperature to remove cell debris, and the supernatant was filtered through 0.45 μm filter. Unused virus stocks were stored at − 80 °C for later use.
P5CS CRISPR/Cas9 knockout in SH-SY5Y cells
For generation of stable cell lines, SH-SY5Y cells were inoculated with lentivirus for 24–48 h, then replenished with fresh culture medium containing the selection drug puromycin at 1 μg/mL. Cells were replenished with fresh culture medium containing puromycin every 2 days until majority of the uninfected control cells were eliminated (in ~ 7–10 days). To generate single cell clones, cells were treated with 1 μg/mL DOX for 24 h and the GFP positive cells were sorted out by flow cytometry. The GFP-positive single cells were then seeded into a 96-well plate and allowed to expand. The cell clones that were depleted of the target protein as analyzed by western blot were used in functional experiments.
Lactate dehydrogenase (LDH) assay
To determine cytotoxicity, LDH release was measured with the LDH Cytotoxicity Assay Kit (Pierce) according to the manufacturer's protocol. 5 × 105 SH-SY5Y cells were plated into 6-well plates and maintained overnight before treatment, following which the cells were treated with METH at various concentrations for 24 h. After treatment, the cell supernatants were collected by centrifugation. Supernatants were assayed for LDH in triplicates as per the manufacturer’s protocol. Absorbances were measured at 490 nm and 680 nm using a spectrophotometer (Biotek). The percentage of cytotoxicity was calculated based on the percentage difference compared with the LDH-positive control provided with the kit.
5 × 105 SH-SY5Y cells were plated in 6-well plate overnight and treated with METH for 24 h. Supernatants were collected, briefly centrifuged to remove cell debris, and analyzed by a Glutamate Colorimetric Assay kit purchased from Cell Biolabs as per the instructions from the manufacturer. For measuring intracellular glutamate, the cells were lysed by sonication in sucrose buffer. After lysis, the cell extracts were centrifuged at 15,000×g for 5 min and the supernatants were subjected to glutamate assay using the kit. All the reactions were performed in triplicates. The glutamate concentrations in the samples were determined by plotting the data against the respective standard curve that was generated in parallel using a series of known glutamate concentrations ranging from 6.25 to 400 µM.
Measurement of intracellular proline levels
5 × 105 SH-SY5Y cells were plated per well in 6-well plates overnight and treated with METH for 24 h. After treatment cells were harvested and lysed in cold PBS with 1% Triton X-100. The cellular debris were removed by centrifugation (10,000×g). The supernatants were transferred to a boiling water bath, and intracellular amino acids were extracted by boiling for 10 min. After centrifugation (5 min, 4 °C, 15,000×g), the supernatant was free of proteins, and intracellular proline was determined as described51,52. Briefly, 100 µL of the supernatant was incubated with 100 µl of acid-ninhydrin (0.25 g ninhydrin dissolved in 6 mL glacial acetic acid and 4 mL 6 M phosphoric acid) and 100 µL of glacial acetic acid for 1 h at 100 °C. The reaction was stopped by incubation on ice for 5 min, and the mixture was extracted with 200 µL toluene. The toluene phase was separated, and absorbance at 520 nm was used to determine the concentration of proline. All the reactions were performed in triplicates and a standard curve ranging in concentrations of 0.05–1 mM proline was generated for determining proline concentration of samples.
Western blot analyses
Cells were treated with various concentrations of METH for 24 h, after which cells were harvested and washed with PBS. Cell lysates were prepared as per our published method50 and quantified according to standard BCA protein assay (Pierce, USA). Equal amounts of cell lysates (20 µg) were resolved on SDS–polyacrylamide gels and transferred to nitrocellulose membranes using a semi-dry blotter (Bio-Rad). Membranes were blocked using Tris-buffered saline with 5% nonfat milk (pH 8.0; Sigma). Blots were then probed with the appropriate primary antibody in blocking buffer overnight at 4 °C. Incubation with secondary anti-mouse or anti-rabbit IgG antibodies conjugated to horseradish peroxidase (1:2000) was performed at room temperature for 1 h. All blots were washed in Tris-buffered saline with Tween 20 (pH 8.0; Sigma) and developed using the enhanced chemiluminescence (ECL) procedure (BioRad). Blots were routinely stripped by Restore Plus Stripping Buffer (Pierce) and reprobed with anti-actin monoclonal antibody to serve as loading controls. The density of the band was evaluated by Bio-Rad imaging-lab 4.0 software (https://www.bio-rad.com/en-pl/product/image-lab-software?ID=KRE6P5E8Z).
Transfection of P5CS expression construct in CHO-K1 cells
CHO-K1 cells (1 × 105) were seeded per well in 6-well plates for 24 h in complete F12K media. Prior to transfection, the cells were pretreated with METH for 1 h and then transfected with pcDNA control vector or P5CS expression vector. Transfections were performed with JetPrime (Polyplus) according to the manufacturer’s instructions. 24 h post transfection the cells were harvested by scraping. The cell supernatants and pellets were collected by centrifugation. Supernatants were assayed for glutamate and the cell pellets were analyzed by western blot as described before.
All protocols were approved by the University of Florida and Vanderbilt University Institutional Animal Care and Use Committee (IACUC) policies and adhered to the NIH guidelines. WT C57BL/6J male mice were obtained from Jackson Laboratories (Vanderbilt; Stock No. 000664) or from the University of Florida Animal Care Services and were maintained on a 12-h light/dark cycle with food and water available ad libitum in their home cages. The mice were injected with a single dose of METH at 2 mg/kg body weight (acute exposure). Control mice received saline injections. The animals were euthanized after 24 h and whole brain were extracted, rapidly rinsed with ice-cold PBS and immediately flash frozen. Punches from the cortical regions were taken for each sample and homogenized manually in a sucrose buffer system supplemented with protease inhibitors. Samples were spun down at high speed for 5 min, the supernatants were transferred into separate tubes and the protein concentration was estimated by BCA assay (Pierce).
For brain slices studies 6-week-old male C57BL/6J mice were euthanized under isoflurane anesthesia after which 300-µm coronal brain slices containing the frontal cortex were prepared from whole brain tissue using a Leica Vibratome in oxygenated (95% O2; 5% CO2) ice-cold artificial cerebrospinal fluid (aCSF, in mM: 119 NaCl, 2.5 KCl, 1.3 MgCl2–6H2O, 2.5 CaCl2–2H2O, 1.0 NaH2PO4–H2O, 26.2 NaHCO3, and 11 glucose). Following a 30-min recovery period, slices were then incubated in either 200 µM amphetamine, 200 µM methamphetamine or vehicle (phosphate buffered saline, PBS). Following a 6-h incubation, cortical enriched tissue was isolated and flash frozen until processing for immunoblot analysis.
Data are expressed as mean ± SEM obtained from at least three independent experiments. Significance of differences between control and treated samples was determined by Student’s t test. Values of p < 0.05 were considered to be statistically significant.
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This work was supported by National Institutes of Health Grants R01 AI136740, R56 AI122960, R24 DA036420, and U54 MD007593 to CD, R03 DA037779, and the Research Centers in Minority Institutions (RCMI) U54MD007586 to JP; and Grants from NIDA/NIH DA026947, DA026947S1, DA043895 to HB. BJ was in part supported by the NIH T32 grant AI007281. BG and CG were supported by NIDA/NIH Grant R01DA040630. This work is also in part supported by the Meharry Translational Research Center (MeTRC) Grant 5U54MD007593-10 and Tennessee CFAR Grant P30 AI110527 from the National Institutes of Health to CD.
The authors declare no competing interests.
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Jones, B., Balasubramaniam, M., Lebowitz, J.J. et al. Activation of proline biosynthesis is critical to maintain glutamate homeostasis during acute methamphetamine exposure. Sci Rep 11, 1422 (2021). https://doi.org/10.1038/s41598-020-80917-7
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