Hyperhomocysteinemia is an emerging comorbidity in ischemic stroke

Abstract

Hyperhomocysteinemia or systemic elevation of the amino acid homocysteine is a common metabolic disorder that is considered to be a risk factor for ischemic stroke. However, it is still unclear whether predisposition to hyperhomocysteinemia could contribute to the severity of stroke outcome. This review highlights the advantages and limitations of the current rodent models of hyperhomocysteinemia, describes the consequence of mild hyperhomocysteinemia on the severity of ischemic brain damage in preclinical studies and summarizes the mechanisms involved in homocysteine induced neurotoxicity. The findings provide the premise for establishing hyperhomocysteinemia as a comorbidity for ischemic stroke and should be taken into consideration while developing potential therapeutic agents for stroke treatment.

Introduction

Hyperhomocysteinemia is a common metabolic disorder characterized by systemic elevation of the thiol amino acid, homocysteine, a potentially toxic metabolite formed as an intermediate of the methionine cycle. Demethylation of dietary methionine leads to the formation of homocysteine in a 3-step process where the enzyme methionine adenosyltransferase (MAT) first catalyzes the transfer of an adenosine group from ATP to methionine resulting in the formation of S-adenosylmethionine (SAM). In the second step, the universal methyl donor SAM transfers its methyl group to acceptor molecules such as DNA, RNA, proteins and lipids using the enzyme methyl transferase (MT), resulting in the formation of S-adenosyl homocysteine (SAH). In the final step, SAH is cleaved by the enzyme S-adenosyl homocysteine hydrolase (SAHH) to produce L-homocysteine (Mato et al., 1997; Mudd and Cantoni, 1958). The plasma concentration of homocysteine is maintained dynamically by either remethylation or transsulfuration pathway (Selhub and Miller, 1992). Remethylation involves recycling of homocysteine to methionine, primarily by the enzyme methionine synthase (MS), with N⁵-methyltetrahydrofolate as the methyl donor and vitamin B12 as an essential cofactor (Leclerc et al., 1998; Weir et al., 1988). In the remethylation pathway tetrahydrofolate derived from dietary folic acid plays a significant role, as it is converted by serine hydroxymethyltransferase (SHMT) to form N⁵,N¹⁰-methylene tetrahydrofolate, which is then reduced by methylene tetrahydrofolate reductase (MTHFR) to generate the methyl donor N⁵-methyltetrahydrofolate (Blom and Smulders, 2011). In some organs, remethylation of homocysteine uses the enzyme betaine-homocysteine methyltransferase (BHMT) to transfer the methyl group from betaine (McKeever et al., 1991; Moretti and Caruso, 2019; Pennypacker et al., 1992). In the transsulfuration pathway, serine is added to homocysteine by the enzyme cystathionine β-synthase (CBS), to form cystathionine, which is then cleaved by cystathionase (CSE) to form cysteine (Ichinohe et al., 2005; Obeid and Herrmann, 2006). Both the enzymes utilize vitamin B6 as an essential cofactor (Obeid et al., 2007) (Fig 1).

Figure 1.

Schematic representation of homocysteine metabolism. MAT: methionine adenosyltransferase; SAM: S-adenosylmethionine; MT: methyl transferase; SAH: S-adenosyl homocysteine; SAHH: S-adenosyl homocysteine hydrolase; DHF: Dihydrofolate; THF: tetrahydrofolate; 5, 10-methyleneTHF: 5, 10-methylene tetrahydrofolate; 5-methyl THF: 5-methyl tetrahydrofolate; MTHFR: N⁵,N¹⁰-methylene tetrahydrofolate reductase; MS: methionine synthase; SHMT: Serine hydroxymethyltransferase; BHMT: betaine-homocysteine methyltranferase; DMG: Dimethylglycine; CBS: cystathionine β-synthase; CSE: cystathionase; TCN2: transcobalamin II; B12: vitamin B12; B6: vitamin B6; GSH: Glutathione; dATP: deoxy-adenosine triphosphate; dTMP: deoxythymidine monophosphate; dUMP: deoxyuridine monophosphate; Pi: inorganic phosphate; PPi: inorganic pyrophosphate.

Several epidemiological studies have indicated that hyperhomocysteinemia may increase the risk for cerebral stroke, potentially due to its impact on venous and arterial atherosclerotic modifications, endothelial dysfunction and vascular inflammation (Austin et al., 2004; Bostom et al., 1999; Hankey and Eikelboom, 2001; Parnetti etal.,2004; Selhub et al., 1995; Shi et al., 2015). For these reasons, in addition to being a risk factor, hyperhomocysteinemia is also considered to be a strong contributor of cardiovascular disease by the World Health Organization (Tinelli et al., 2019). Analysis of multiple prospective and retrospective studies has shown about 19% reduction in the risk for stroke for every 3 μM decrease in plasma homocysteine concentration with vitamin supplementation (Hankey, 2006; Homocysteine Studies, 2002). As such, increased homocysteine levels have been proposed to be used as a preclinical biomarker for stroke (Kelly and Furie, 2002; Rehman et al., 2020; Shi et al., 2018). However, large intervention studies with folate supplementation to reduce homocysteine levels produced conflicting data, raising some concerns, especially about the size of the population evaluated in the earlier studies, which showed positive outcome (Chrysant and Chrysant, 2018; Herrmann et al., 2007).

Stroke is the second leading cause of death and a major cause of disability worldwide with an annual mortality rate of 5.5 million people and chronic disability among 50% of the survivors (Donkor, 2018; Writing Group et al., 2016). Stroke in most cases is caused due to an abrupt blockage of blood vessel and reduction in blood supply to the brain (ischemic stroke), but in some instances may be caused due to the rupture of a blood vessel (hemorrhagic stroke) causing bleeding in the brain or the subarachnoid space (Amarenco et al., 2009; Donnan et al., 2008). Treatment options for stroke are still limited. In acute ischemic stroke reperfusion with tissue plasminogen activator and surgical recanalization in a small number of cases are the only pharmacologic therapy that is available. The complexity of this clinical condition has been attributed to the prevalence of multiple co-morbid factors, which include hypertension, hyperlipidemia, diabetes, atrial fibrillation and advanced age (Fischer et al., 2006; Gallacher et al., 2014; Guthrie et al., 2012; Karatepe et al., 2008). Emerging preclinical studies now indicate that mild hyperhomocysteinemia could also worsen stroke outcome, which provides the basis to consider hyperhomocysteinemia as a comorbidity that could directly impact the pathophysiology of stroke outcome. The purpose of this review is to summarize the outcome of these recent preclinical studies that have evaluated the effects of predisposition to mild hyperhomocysteinemia on the severity of stroke outcome. The review will further outline the current concepts in the molecular mechanisms of homocysteine mediated neurotoxicity that could be targeted for the development of therapeutic intervention in ischemic stroke.

Causes of hyperhomocysteinemia

The total plasma homocysteine levels in the general human population ranges between 7-14 μM (Austin et al., 2004; Hankey and Eikelboom, 2001; Obeid and Herrmann, 2006). However, nutritional deficiency of dietary folate, vitamin B12 and vitamin B6 results in mild (16 – 30 μM) to moderate (30 – 100 μM) increases in total plasma homocysteine levels. Mild to moderate hyperhomocysteinemia can occur following consumption of alcohol, tobacco, coffee or various drugs that interfere with methionine metabolism. Sedentary lifestyle, old age and menopause are also determinants of hyperhomocysteinemia. In addition, moderate hyperhomocysteinemia can occur due to genetic mutation and altered functioning of the catalytic enzymes involved in homocysteine remethylation, which includes MTHFR (MTHFR gene), MS (MTR gene), methionine synthase reductase (MTRR gene), BHMT (BHMT gene) and serine hydroxymethyltransferase 1 (SHMT1 gene) (van Meurs et al., 2013). In MTHFR gene, over 30 point-mutations have been identified so far, of which C677T and A1298C seem to have the most clinical significance (Rozen, 1997; Zhou et al., 2014). The missense mutation C677T, involving substitution of cytosine at position 677 to thymine is the most frequently studied genetic polymorphism in MTHFR, which results in the conversion of alanine to valine. It has been shown to reduce MTHFR enzyme activity by 70% and 35% for homozygous and heterozygous mutations respectively (Frosst et al., 1995; Tsang et al., 2015). The A1298C mutation in the MTHFR gene results in substitution of glutamate with alanine and also reduces enzyme activity by 50-60% (van der Put et al., 1998; Weisberg et al., 1998). In some populations, A1298C polymorphism has been associated with higher homocysteine concentration and decreased plasma folate (Kumar et al., 2005; Lalouschek et al., 1999; Malinow et al., 1997; McCarthy et al., 2004; Narayanan et al., 2004; Press et al., 1999; Weisberg et al., 1998). Mutations in the MTR gene, although less common, has also been associated with hyperhomocysteinemia. It has at least 2028 single nucleotide polymorphisms (SNPs) and among these SNPs, the conversion of aspartic acid to glycine residue at position 2756 (A2756G) in the open reading frame results in decreased enzymatic activity that contributes to moderate elevation of homocysteine (Harmon et al., 1999; Wilson et al., 1998; Wu et al., 2017). In addition, several polymorphisms in genes of enzymes involved in folate recycling (MTRR and SHMT1) and transportation of vitamin B12 (transcobalamin II or TCN2) have also been reported to be involved in moderate increase in plasma homocysteine (Low et al., 2011). Severe hyperhomocysteinemic conditions (100-500 μM) are caused by homozygous deficiency of CBS enzyme, while the heterozygous form of the enzyme is characterized with normal levels of homocysteine (Kruger et al., 2000). Amongst the 164 different mutations and polymorphisms in the CBS gene, T833C, where isoleucine is mutated to threonine at position 278 (I278T) has been reported in virtually every studied populations around the world while others only occur in specific populations (de Leon Bautista et al., 2018; Kraus, 1998). Such severe hyperhomocysteinemic conditions, which is accompanied with homocystinuria result in multi-system disorders with cardiovascular and neurological dysfunction and increased mortality when left untreated (Austin et al., 2004; Hankey and Eikelboom, 2001; Obeid and Herrmann, 2006; Zoccolella et al., 2006). The most prevalent genetic polymorphisms for the enzymes associated with homocysteine metabolism are listed in Table 1.

Table 1:

Common polymorphisms in genes associated with homocysteine metabolism that elevate plasma homocysteine level

Pathway	Enzyme	Gene	Common SNPs	Amino acid codon change
Remethylation	Methyl tetrahydrofolate reductase (MTHFR)	MTHFR gene	rs1801133 (C677T) rs1801131 (A1298C)	A > V E > A P > P
	Methionine synthase (MS)	MTR gene	rs2066470	D > G
		BHMT gene	rs1805087 (A2756G)	G > A
	Betaine-homocysteine methyltransferase (BHMT)		rs3733890
Vitamin B12 recycling	Methionine synthase reductase (MTRR)	MTRR gene	rs1801394 (A66G) rs10380	I > M H > Y
Vitamin B12 transport	Transcobalamin II (TCN2)	TCN2 gene	rs1801198 (G776C)	P > R
Folate recycling	Serine hydroxy-methyltransferase 1 SHMT1	SHMT1 gene	rs1979277 (C1420T)	L > F
Transsulfuration	Cystathionine β – synthase (CBS)	CBS gene	rs234706 (C699T) rs2298758 (T833C) rs1801181	Y > Y I > T A > A

Numerous studies have demonstrated that the disruption of homocysteine metabolism leads to hyperhomocysteinemia, which increases the risk factor for several complex disorders involving cardiovascular, renal, gastrointestinal and central nervous systems (Al Mutairi, 2020; Ientile et al., 2010). In the central nervous system such disorders include cognitive impairment, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, epilepsy and cerebral stroke (Austin et al., 2004; Hankey and Eikelboom, 2001; Obeid and Herrmann, 2006; Zoccolella et al., 2006). To reduce homocysteine related disorders in the USA, the FDA mandated fortification of food with folic acid (Federal Register 61:8752, 1996). In spite of this preventive measure, the incidence of hyperhomocysteinemia in the elderly population is significantly large due to low nutritional absorption and decreased metabolic function with advanced age, insufficient renal / hepatic function or medications (vitamin B6 and folate antagonists) (Austin et al., 2004; Hankey and Eikelboom, 2001; McCully, 2007; Obeid and Herrmann, 2006; Zoccolella et al., 2006). Depending on gender differences, 44-46% of the elderly population (60 years and older) in the USA is still found to be hyperhomocysteinemic (Carmel et al., 1999; Green, 1995; Janson et al., 2002; Ramos et al., 2005; Robles et al., 2005). Notably, the prevalence of hyperhomocysteinemia outside the USA is even higher (66–76%) (Janson et al., 2002; Selhub et al., 1999). Although extensive studies have investigated the role of hyperhomocysteinemia as a risk factor for multiple neurological disorders (Hankey and Eikelboom, 2001; Parnetti et al., 2004; Seshadri et al., 2002; Zoccolella et al., 2010; Zoccolella et al., 2006), only few studies have begun to explore whether pre-disposition to hyperhomocysteinemia could contribute to the severity of these neurological disorders.

Animal models of hyperhomocysteinemia

In the last three decades, multiple murine models of hyperhomocysteinemia have been developed, which has helped considerably in our understanding of this metabolic condition. Hyperhomocysteinemia has been induced in both rat and mice through dietary modifications that involve either (1) adding excess methionine to the diet, (2) a diet deficient in folate, vitamin B12 and/or vitamin B6 or (3) a diet deficient in choline (Dayal and Lentz, 2008; Duan et al., 2002; Setoue et al., 2008; Tan et al., 2006; Vitvitsky et al., 2004). In some animal models the above dietary components have also been combined to ensure higher levels of total plasma homocysteine (Dayal et al., 2005; Dayal and Lentz, 2008; Devlin et al., 2004; Vitvitsky et al., 2004). In all of these diet-induced animal models, the levels of total plasma homocysteine increase moderately, ranging between 18 to 60 μM (Dayal and Lentz, 2008). The severity of hyperhomocysteinemia achieved in these models depends on the extent and duration of methionine supplementation and deficiency in folic acid, vitamin B12, vitamin B6 and choline in the diet as well as the number of dietary components modified.

In addition to dietary modifications, various genetic models of hyperhomocysteinemia have been developed in mice. Targeted disruption of genes encoding for the MTHFR, MS and CBS enzymes result in defective homocysteine metabolism resulting in hyperhomocysteinemia (Chen et al., 2001; Swanson et al., 2001; Watanabe et al., 1995). The total plasma homocysteine levels observed in these genetic models varies with the type of gene deleted. The MTHFR knockout (MTHFR−/−) mice on a normal diet develop moderate hyperhomocysteinemia with total plasma homocysteine of approximately 30 μM (Chen et al., 2001). However, these MTHFR−/− mice have increased mortality beyond 5 weeks of age accompanied by cerebellar hypoplasia, and both conditions can be improved with dietary supplementation with betaine during pregnancy and lactation (Chen et al., 2001). The MS knockout mice (MTR−/−) are embryonically lethal and dietary supplementation with methionine, folic acid, betaine or choline does not increase their survival rate (Swanson et al., 2001). The CBS knockout mice (CBS−/−) have total plasma homocysteine level as high as 200 μM, which is accompanied by retarded growth, fatty liver and vascular pathology (Watanabe et al., 1995). They also have poor survival rate beyond 3 to 5 weeks of age and supplementation of choline-enriched diet may marginally improve early mortality (Watanabe et al., 1995). Due to the high mortality of the above genetic models, many studies have utilized MTHFR, MS and CBS heterozygous mice subjected to dietary modifications to elevate their total plasma homocysteine (Dayal et al., 2005; Devlin et al., 2004; Virdis et al., 2003; Watanabe et al., 1995). Other approaches involving conditional knockout or single point mutations of these genes have also been implemented in an attempt to overcome these mortality issues (Cheng et al., 2005; Wang et al., 2004).

Although, it is clearly evident that the MTHFR, MS and CBS enzymes are important to maintain homeostasis of homocysteine metabolism, they also play critical roles in multiple aspects of development and maturation of fetus and early life (Chen et al., 2001; Swanson et al., 2001; Watanabe et al., 1995). Similarly, many of the dietary components involved in homocysteine metabolism also have critical roles in regulating various other physiological processes. Therefore, a major limitation of the above animal models is that in addition to elevation of plasma homocysteine, there are alterations in other metabolic pathways. As such, the pathophysiological outcome observed in these models cannot be attributed to hyperhomocysteinemia alone (Dayal and Lentz, 2008; Endres et al., 2005). This issue has been highlighted in an earlier study where folate deficient mice subjected to ischemia/reperfusion injury showed a significant increase in brain damage, when compared to mice fed with normal diet (Endres et al., 2005). Although these animals had higher total plasma homocysteine levels, the authors emphasized that their findings could not differentiate between lack of folate or increased homocysteine levels as the potential cause of increased vulnerability to stroke. According to their findings, the increased susceptibility to ischemic insult in folate deficient mice is attributed to uracil mis-incorporation and inefficient uracil base excision repair of DNA, both of which are folic acid dependent processes.

To understand the pathophysiology of hyperhomocysteinemia, it is therefore critically important to develop animal models where the pathological outcome could be directly attributed to the elevated level of plasma homocysteine. To address this issue, some studies have started to generate hyperhomocysteinemic animal models by implanting osmotic pumps containing homocysteine to provide slow and continuous systemic infusion of the amino acid, allowing direct evaluation of the detrimental effects of hyperhomocysteinemia (Chen et al., 2002; Christie et al., 2005; Jindal et al., 2019). Such osmotic infusion of L-homocysteine in mice and rats could increase total plasma homocysteine within 3 days of pump implantation (Jindal et al., 2019).

Predisposition to hyperhomocysteinemia exacerbates ischemic brain injury

Earlier studies have demonstrated that treatment of neuronal cultures with homocysteine leads to dose-dependent increase in neuronal cell death (Kruman et al., 2000; Lipton et al., 1997). Interestingly, the study by Kruman et al. (2000) further demonstrated that treatment of neuronal cultures with homocysteine in conjunction with glutamate leads to significant increase in neuronal cell death, when compared to treatment with homocysteine or glutamate alone. These findings provided the first indication that homocysteine-induced neurotoxicity could be distinctly different from glutamate mediated excitotoxic neuronal injury. Since, excessive release of the neurotransmitter glutamate is the key initiating factor leading to neurodegeneration following ischemic stroke, more recent studies have started to investigate the effect of elevated levels of homocysteine during an ischemic insult. Using oxygen glucose deprivation (OGD) as an in vitro model of ischemic stroke, it has been showed that OGD (2h) in the presence of L-homocysteine (50 μM) leads to two-fold increase in neuronal cell death, as compared to L-homocysteine or OGD treatment alone (Jindal et al., 2019). The study by Jindal et al. (2019) also generated rat and mice models of mild hyperhomocysteinemia by osmotic infusion of L-homocysteine, to investigate the effect of elevated plasma homocysteine on stroke outcome. Their findings show that transient ischemic insult by middle cerebral artery occlusion (MCAO) followed by reperfusion resulted in substantial increase in mortality in the hyperhomocysteinemic animals when compared to the stroke outcome in the control animals with normal total plasma homocysteine. In the surviving hyperhomocysteinemic animals, evaluation of infarct size by magnetic resonance imaging (MRI) showed significant increase in ischemic lesion volume 24 h after the ischemic insult when compared to the lesion volume in the control group with normal plasma homocysteine level. Longitudinal evaluation showed significant loss in structural integrity within the infarcted area under hyperhomocysteinemic conditions 14 days after ischemic insult. In addition, significant deficits in sensorimotor functions and behavioral outcomes was observed in the hyperhomocysteinemic animals. The exacerbation of ischemic brain injury and higher mortality rate in the hyperhomocysteinemic animals demonstrated for the first time a robust and direct effect of elevated plasma homocysteine on stroke outcome. Another recent study also demonstrated that mild hyperhomocysteinemia in heterozygous CBS deficient mice, expressing a zinc inducible mutant human CBS transgene, could also lead to exacerbation of ischemic brain injury (Gu et al., 2020). The exacerbation of ischemic brain injury observed in these hyperhomocysteinemic animal models suggests that post-stroke disruption of blood brain barrier leads to rapid increase in brain homocysteine level resulting in additional neurotoxicity. It is unlikely that interventions to reduce plasma homocysteine level as a post-stroke treatment paradigm would have any effect in mitigating the severity of ischemic brain damage under hyperhomocysteinemic condition. As such, a systematic evaluation of the underlying mechanisms associated with homocysteine induced neurotoxicity and its correlation with ischemic events is essential for development of potential therapeutic interventions to counteract the devastating effect of hyperhomocysteinemia on stroke outcome.

Mechanistic basis of hyperhomocysteinemia-induced exacerbation of ischemic brain damage

Homocysteine is a known agonist of N-methyl-D-aspartate receptors (NMDAR) (Lipton et al., 1997). It has been demonstrated that homocysteine acts as an agonist at the glutamate site of NMDAR under high concentrations of glycine (50 μM). In this context, additional studies have shown that disruption of the blood brain barrier following stroke (Palmer et al., 1994) or head trauma (Phillis et al., 1994) raises glycine level in the cortex from ~1 μM to ~100 μM. Blood brain barrier damage also leads to an increase in homocysteine level in the cortex from ~10 μM to plasma levels of homocysteine for prolonged time periods. (Lipton et al., 1997). These findings imply that ischemic stroke under hyperhomocysteinemic conditions could lead to elevated homocysteine and glycine levels in the brain, whose synergistic action results in NMDAR mediated neurotoxicity. Consistent with this interpretation several studies using mixed neuronal and glial culture, hippocampal neuronal culture and cortical neuronal culture have demonstrated that homocysteine-induced neurotoxicity is mediated through NMDAR stimulation (Deep et al., 2019; Kruman et al., 2000; Lipton et al., 1997; Poddar and Paul, 2009, 2013).

Functional NMDARs in the adult forebrain are composed of heteromeric assemblies of GluN1/GluN2A, GluN1/GluN2B or GluN1/GluN2A/GluN2B subunits. It has been proposed that the subunit composition of NMDARs determines their function under different pathophysiological conditions (Hardingham et al., 2002; Li et al., 2002; Zhang et al., 2007). GluN2A-containing NMDARs (GluN2A-NMDAR) have been shown to promote synaptic plasticity including long-term potentiation and spatial memory formation (Dingledine et al., 1999; Hardingham and Bading, 2003; Lynch and Guttmann, 2002; Scimemi et al., 2004; Vicini et al., 1998). Studies using glutamate as an agonist for NMDAR showed that growth and survival-promoting signals are derived primarily from GluN2A-NMDAR stimulation (Liu et al., 2007). In contrast, GluN2B containing NMDARs (GluN2B-NMDAR) are involved in long-term depression (Dalton et al., 2012; Foster et al., 2010; Fox et al., 2006; Li et al., 2006; Liu et al., 2004; Massey et al., 2004). In addition, death-promoting signal have been shown to be generally triggered by excessive release of the neurotransmitter glutamate leading to over activation of GluN2B-NMDAR (Chen et al., 2008; Hardingham et al., 2002; Li et al., 2002; Liu et al., 2007; Zhang et al., 2007). Consistent with these findings, several studies have demonstrated that selective inhibition of GluN2A-NMDAR either has no effect or exacerbate neuronal death, whereas selective inhibition of GluN2B-NMDAR is neuroprotective (Chen et al., 2008; DeRidder et al., 2006; Lai et al., 2011; Liu et al., 2007; Morikawa et al., 1998; Schumann et al., 2008).

Since glutamate and homocysteine are both NMDAR agonist, it has been assumed that they activate GluN2B-NMDAR mediated intracellular pathways leading to neurotoxicity, thereby limiting studies in this area for the past two decades. However, emerging evidence indicate that in contrast to glutamate, homocysteine-induced neurotoxicity involves GluN2A-NMDAR stimulation. Using pharmacological inhibitors for GluN2B-NMDAR in cortical neuronal cultures exposed to homocysteine, an earlier study showed that homocysteine-NMDAR induced neurotoxicity is independent of GluN2B-NMDAR stimulation (Poddar and Paul, 2009). Additionally, a more recent study, using pharmacological inhibitors for both GluN2A-NMDAR (NVP-AAM077) and GluN2B-NMDAR (Ro 25-6981) as well as neuronal cultures from GluN2A-subunit knockout (GluN2A-KO) mice established directly the role of GluN2A-NMDAR in homocysteine-induced neurotoxicity (Deep et al., 2019). These findings indicate that GluN2A-NMDAR mediated intracellular signaling in neurons following exposure to glutamate and homocysteine are distinctly different and a critical determinant of whether neurons survive or die. Multiple studies have now shown that the magnitude of Ca²⁺ influx through NMDAR in response to glutamate or NMDA is significantly higher than that observed with homocysteine (Deep et al., 2019; Kruman et al., 2000; Lipton et al., 1997; Sibarov et al., 2016). Following exposure to glutamate (50 μM) intracellular Ca²⁺ in neurons increases rapidly but transiently reaching a peak within 2.5 min (~21 fold) and then decline progressively to basal level within 10 min (Connor et al., 1988; de Erausquin et al., 1990; Deep et al., 2019; Glaum et al., 1990; Randall and Thayer, 1992). In contrast, exposure to L-homocysteine (50 μM) leads to a slow and progressive increase in intracellular Ca²⁺ resulting in a ~7-fold increase by 60 min as compared to unstimulated control cells (Deep et al., 2019; Kruman et al., 2000; Sibarov et al., 2016). The study by Deep et al (2019) utilized pharmacological inhibitors of GluN2A- and GluN2B-NMDARs as well as neuronal cultures from GluN2A-KO mice to establish the role of GluN2A-NMDARs in homocysteine-induced sustained Ca²⁺ influx in neurons. Whereas, the study by Sibarov et al. (2016), using patch clamp recordings in primary neuronal cell cultures, further showed that steady state currents generated with DL-homocysteine (100 μM) is resistant to ifenprodil, a selective inhibitor of GluN2B-NMDAR, indicating that the current generated was primarily GluN2A-NMDAR dependent (Sibarov et al., 2016). The authors confirmed their findings in HEK293 cells co-expressing GluN1 and GluN2A (GluN1/2A) or GluN1 and GluN2B (GluN1/2B) NMDAR subunits.

An additional study investigated the role of GluN2A-NMDARs in the exacerbation of ischemic brain injury under hyperhomocysteinemic conditions (Jindal et al., 2019). An important finding from this study is that pharmacological inhibition of GluN2A-NMDAR with NVP-AAM077 considerably reduces the extent of mortality observed in hyperhomocysteinemic rats subjected to ischemia-reperfusion injury. GluN2A-NMDAR inhibition in hyperhomocysteinemic rats also resulted in significant reduction in post-ischemic infarct size and behavioral deficits observed 24h after reperfusion. In contrast, inhibition of GluN2B-NMDAR with Ro 25-6981 failed to significantly reduce infarct size observed in hyperhomocysteinemic rats, indicating that GluN2B-NMDAR stimulation does not play a role in mediating ischemic injury under hyperhomocysteinemic conditions (Jindal et al., 2019). Interestingly, GluN2A-NMDAR inhibition did not alter the extent of ischemic injury in rats with normal plasma homocysteine level, which is consistent with an earlier finding (Liu et al., 2007). However, GluN2B-NMDAR significantly reduced ischemic injury in control rats (Jindal et al., 2019). This later finding is also consistent with earlier reports indicating that GluN2B-NMDAR stimulation is the primary mediator of ischemia induced brain damage (Chen et al., 2008; Lai et al., 2011; Liu et al., 2007; Wu and Tymianski, 2018). Further validation for a role of GluN2A-NMDARs in the exacerbation of ischemic brain injury under hyperhomocysteinemic condition came from additional studies in GluN2A-KO mice, where in the absence of functional GluN2A-NMDARs, ischemic lesion volume did not increase with elevated levels of total plasma homocysteine (Jindal et al., 2019).

Collectively, these studies indicate that hyperhomocysteinemia triggers unique intracellular signaling pathways mediated through GluN2A-NMDAR stimulation that act in conjunction with ischemia-induced pathways involving GluN2B-NMDAR stimulation to enhance the pathology of stroke under hyperhomocysteinemic conditions (Fig. 2). Studies on the cellular mechanisms of NMDAR-mediated neuronal injury revealed that glutamate-mediated NMDAR stimulation leads to transient increase in phosphorylation and activation of extracellular-signal regulated mitogen activated protein kinase (ERK MAPK), whereas homocysteine-mediated NMDAR stimulation leads to sustained phosphorylation and activation of ERK MAPK (Chandler et al., 2001; Jiang et al., 2000a, b; Paul et al., 2003; Poddar and Paul, 2009, 2013). Several studies have also shown that the glutamate-NMDAR induced transient increase in ERK MAPK activation plays an important role in neuronal maturation, survival and long-term potentiation (Atkins et al., 1998; English and Sweatt, 1997; Xia et al., 1995). In contrast, homocysteine-NMDAR induced sustained activation of ERK MAPK contributes to neuronal cell death (Deep et al., 2019; Poddar and Paul, 2009, 2013). Additional studies using pharmacological and knockdown approaches further revealed that homocysteine-induced sustained increase in ERK MAPK phosphorylation is Ca²⁺ dependent and involves stimulation of GluN2A-NMDARs (Deep et al., 2019; Jindal et al., 2019; Poddar and Paul, 2009, 2013). Collectively, these findings imply that the duration of ERK MAP kinase signaling in neurons following NMDAR stimulation depends on the extracellular stimulus leading to different cellular responses. Studies in both ex vivo (OGD) and in vivo (MCAO) models of ischemia have also evaluated the temporal profile of ERK MAPK phosphorylation in the presence of homocysteine. Ex vivo experiments in neuronal cultures subjected to OGD in the presence of elevated levels of homocysteine showed a sustained increase in ERK MAPK phosphorylation throughout the duration of experiment, as compared to the rapid and transient activation of ERK MAPK with OGD alone (Jindal et al., 2019). Ischemic insult (MCAO, 60 min) in hyperhomocysteinemic rats also induced sustained increase in ERK MAPK during reperfusion (Jindal et al., 2019). These studies also demonstrated a role of GluN2A-NMDAR in ERK MAPK phosphorylation, confirming GluN2A-NMDAR mediated signaling in triggering detrimental pathways under hyperhomocysteinemic conditions (Fig. 2).

Figure 2.

Signaling mechanisms leading to exacerbation of ischemic brain injury under (A) normal and (B) hyperhomocysteinemic conditions.

The signaling cascades downstream of ERK MAPK activation that is involved in homocysteine-GluN2A-NMDAR induced neurotoxicity is still being investigated. Emerging evidence indicate that homocysteine-GluN2A-NMDAR dependent ERK MAPK activation leads to decrease in surface expression of GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. As a result, Ca²⁺ influx through the GluA2-lacking Ca²⁺ permeable AMPA receptors leads to p38 MAPK activation (Poddar et al., 2017; Poddar and Paul, 2013). This novel crosstalk between ERK and p38 MAPK plays a key role in homocysteine-induced neurotoxicity and subsequent progression of ischemic brain damage. A schematic representation of homocysteine-GluN2A NMDAR mediated intracellular signaling pathway is presented in Figure 3.

Figure 3.

Intracellular-signaling pathways involved in homocysteine induced neuronal cell death.

Conclusion and future challenges

Hyperhomocysteinemia or chronic elevation of plasma homocysteine is widely recognized as a risk factor for human ischemic stroke. However, it still remains unclear whether elevated levels of plasma homocysteine could directly contribute to the severity of stroke outcome. This review highlights the novel findings from emerging studies where the experimental paradigm combined ischemic insult with elevated homocysteine level in both ex vivo and in vivo studies to evaluate the influence of hyperhomocysteinemia on ischemic injury in neurons and in the brain. These exciting new studies show that homocysteine-mediated GluN2A-NMDAR signaling is a critical determinant of the enhanced neurotoxicity and brain damage observed when both the stressors were combined. The studies also emphasize the role of sustained ERK MAPK activation in ischemic cell death under hyperhomocysteinemic condition. As GluN2A-NMDARs are generally thought to promote neuronal survival, these novel finding implying a role of GluN2A-NMDARs in neurotoxicity could lead to a major paradigm shift in the field of NMDAR signaling. Extensive evaluation of the signaling cascade downstream of ERK MAPK activation is also important for the development of potential therapeutic targets that could mitigate the detrimental effects of homocysteine-mediated GluN2A-NMDAR stimulation.

With the prevalence of hyperhomocysteinemia in the general population, there is an urgent need for more studies in clinically relevant stroke models and human stroke patients to further validate the role of hyperhomocysteinemia as a predictive biomarker for the severity of stroke outcome. Recognition of hyperhomocysteinemia as a comorbid factor would further help in exploring novel therapeutic targets downstream of GluN2A-NMDAR signaling. Such therapies, when combined with thrombolytic agents, could eventually reduce the severity of ischemic brain damage in individuals suffering from hyperhomocysteinemia. In the meantime, for stroke treatment in humans predisposed to hyperhomocysteinemia, strategies to reduce plasma homocysteine after an ischemic insult may not be a suitable therapeutic approach but could still be effective in repair mechanisms or as a precaution for recurrence of stroke.