New Perspectives for Developing Therapeutic Bioconjugates of Metabolite-Depleting Enzymes: Lessons Learned Combating Glutamate Excitotoxicity.

Glutamate, the main excitatory neurotransmitter in the central nervous system, plays an essential role in several cognitive activities such as memorizing and learning. Excessive glutamate release and disturbance of glutamate homeostasis participates in multiple neuronal pathologies including cerebral ischemia (inadequate blood supply), traumatic brain injury (e.g., from a fall or an accident), multiple sclerosis, epilepsy, migraine, fetal hypoxia, or Alzheimer's disease. Attenuating excitotoxicity by, for example, targeting glutamate receptors has proved to be beneficial in animal models but has largely failed in clinical trials because of toxic side effects. New therapeutic concepts have been explored to reduce the excitotoxic effect caused by the excessive glutamate release by using or stimulating glutamate-depleting enzymes in the bloodstream. These enzymes indirectly act upon the brain by depleting glutamate in the bloodstream, which is believed to siphon it out of the brain. Recent studies have shown that bioconjugate approaches applied to such enzymes exacerbate this therapeutic effect but raise additional questions for future research. This Perspective provides an overview of lessons learned by our group when exploring bioconjugate approaches for combatting glutamate excitotoxicity as an illustration of how research on therapeutic bioconjugates is evolving.

Associations between RNA-Binding Motif Protein 3, Fibroblast Growth Factor 21, and Clinical Outcome in Patients with Stroke.

BACKGROUND: RNA-binding motif protein 3 (RBM3) is a cold-induced marker of good functional outcome of ischemic stroke that is promising as a protective target. Fibroblast growth factor 21 (FGF21) is an obesity- and temperature-related hormone that upregulates the expression of RBM3, which is beneficial as a recombinant treatment and has been tested under different experimental pathological conditions, including stroke. However, the interaction between RBM3 and FGF21 has not yet been tested for clinical stroke conditions. METHODS: In a sample of 66 stroke patients, we analyzed the associations between the FGF21 and RBM3 serum concentrations on admission and at 72 h, body weight, maximum temperature during the first 24 h, and the outcome of patients at 3 months. We also analyzed their association with biomarkers of obesity (adiponectin and leptin) and inflammation (interleukin-6 (IL-6) and interleukin (IL-10)). RESULTS: Higher concentrations of FGF21 on admission and RBM3 at 72 h were associated with good outcomes. Serum FGF21 and RBM3 were directly related to body mass index and inversely related to the maximum temperature during the first 24 h. We found a positive association between the FGF21 concentrations in obese patients with leptin and a negative correlation with adiponectin in non-obese participants. CONCLUSIONS: This clinical study demonstrates the association between RBM3 and FGF21 levels and the outcome of stroke patients. Although further investigations are required, these data support the pharmacological induction of RBM3 as a promising protective therapy.

Characterization of a Temporal Profile of Biomarkers as an Index for Ischemic Stroke Onset Definition.

BACKGROUND AND PURPOSE: Stroke is a dynamic process in terms of molecular mechanisms, with prominent glutamate-mediated excitotoxicity at the onset of symptoms followed by IL-6-mediated inflammation. Our aim was to study a serum glutamate/IL-6 ratio as an index for stroke onset definition. METHODS: A total of 4408 ischemic stroke patients were recruited and then subdivided into four quartiles according to latency time in minutes (0-121, 121-185, 185-277 and >277). Latency time is defined as the time between stroke onset and treatment at the neurological unit. The primary endpoint of the study was the association of early latency times with different clinical aspects and serum markers. Serum glutamate and interleukin-6 (IL-6) levels at admission were selected as the main markers for excitotoxicity and inflammation, respectively. RESULTS: Glutamate serum levels were significantly higher in the earlier latency time compared with the higher latency times (p < 0.0001). IL-6 levels were lower in early latency times (p < 0.0001). Patients with a glutamate/IL-6 index on admission of >5 were associated with a latency time of <121 min from the onset of symptoms with a sensitivity of 88% and a specificity of 80%. CONCLUSIONS: The glutamate/IL-6 index allows the development of a ratio for an easy, non-invasive early identification of the onset of ischemic stroke symptoms, thus offering a new tool for selecting early stroke patient candidates for reperfusion therapies.

Inhibition of endogenous blood glutamate oxaloacetate transaminase enhances the ischemic damage.

Glutamate oxaloacetate transaminase 1 (GOT1) enzyme plays a critical role in the cell metabolism by participating in the carbohydrate and amino acid metabolism. In ischemic stroke, we have demonstrated that recombinant GOT1 acts as a novel neuroprotective treatment against the excess of extracellular glutamate that accumulates in the brain following ischemic stroke. In this study, we investigated the inhibitory effect of GOT1 on brain metabolism and on the ischemic damage in a rat model of ischemic stroke by means of a specific antibody developed against this enzyme. Inhibition of GOT1 caused higher brain glutamate and lactate levels and this response was associated with larger ischemic lesion. This study represents the first demonstration that the inhibition of the blood GOT1 activity leads to more severe ischemic damage and poorer outcome and supports the protective role of GOT1 against ischemic insults.

Sustained blood glutamate scavenging enhances protection in ischemic stroke.

Stroke is a major cause of morbidity, mortality, and disability. During ischemic stroke, a marked and prolonged rise of glutamate concentration in the brain causes neuronal cell death. This study explores the protective effect of a bioconjugate form of glutamate oxaloacetate transaminase (hrGOT), which catalyzes the depletion of blood glutamate in the bloodstream for ~6 days following a single administration. When treated with this bioconjugate, a significant reduction of the infarct volume and a better retention of sensorimotor function was observed for ischemic rats compared to those treated with saline. Moreover, the equivalent dose of native hrGOT yielded similar results to the saline treated group for some tests. Targeting the bioconjugate to the blood-brain-barrier did not improve its performance. The data suggest that the bioconjugates draw glutamate out of the brain by displacing homeostasis between the different glutamate pools of the body.

Cold stress protein RBM3 responds to hypothermia and is associated with good stroke outcome.

RNA-binding motif protein 3 is a molecular marker of hypothermia that has proved neuroprotective in neurodegenerative disease models. However, its relationship to the well-recognized therapeutic effect of hypothermia in ischaemic stroke had not been studied. In this work, the expression of RNA-binding motif protein 3 was investigated in ischaemic animal models subjected to systemic and focal brain hypothermia, specifically the effects of RNA-binding motif protein 3 silencing and overexpression on ischaemic lesions. Moreover, the association of RNA-binding motif protein 3 levels with body temperature and clinical outcome was evaluated in two independent cohorts of acute ischaemic stroke patients (n = 215); these levels were also determined in a third cohort of 31 patients derived from the phase III EuroHYP-1 trial of therapeutic cooling in ischaemic stroke. The preclinical data confirmed the increase of brain RNA-binding motif protein 3 levels in ischaemic animals subjected to systemic and focal hypothermia; this increase was selectively higher in the cooled hemisphere of animals undergoing focal brain hypothermia, thus confirming the direct effect of hypothermia on RNA-binding motif protein 3 expression, while RNA-binding motif protein 3 up-regulation in ischaemic brain regions led to functional recovery. Clinically, patients with body temperature <37.5°C in the first two cohorts had higher RNA-binding motif protein 3 values at 24 h and good outcome at 3 months post-ischaemic stroke, while RNA-binding motif protein 3 levels in the cooled third cohort tended to exceed those in placebo-treated patients. These results make RNA-binding motif protein 3 a molecular marker associated with the effect of hypothermia in ischaemic stroke and suggest its potential application as a promising protective target.

Blood glutamate EAAT2-cell grabbing therapy in cerebral ischemia.

BACKGROUND: Excitatory amino acid transporter 2 (EAAT2) plays a pivotal role in glutamate clearance in the adult brain, thereby preventing excitotoxic effects. Considering the high efficacy of EAAT2 for glutamate uptake, we hypothesized that the expression of this transporter in mesenchymal stem cells (MSCs) for systemic administration could yield a cell-based glutamate-grabbing therapy, combining the intrinsic properties of these cells with excitotoxic protection. METHODS: To address this hypothesis, EAAT2-encoding cDNA was introduced into MSCs and human embryonic kidney 293 cells (HEK cells) as the control cell line. EAAT2 expression and functionality were evaluated by in vitro assays. Blood glutamate-grabbing activity was tested in healthy and ischemic rat models treated with 3 × 106 and 9 × 106 cells/animal. FINDINGS: The expression of EAAT2 in both cell types conferred the expected glutamate-grabbing activity in in vitro and in vivo studies. The functional improvement observed in ischemic rats treated with EAAT2-HEK at low dose, confirmed that this effect was indeed mediated by the glutamate-grabbing activity associated with EAAT2 functionality. Unexpectedly, both cell doses of non-transfected MSCs induced higher protection than transfected EAAT2-MSCs by another mechanism independent of the glutamate-grabbing capacity. INTERPRETATION: Although the transfection procedure most likely interferes with some of the intrinsic protective mechanisms of mesenchymal cells, the results show that the induced expression of EAAT2 in cells represents a novel alternative to mitigate the excitotoxic effects of glutamate and paves the way to combine this strategy with current cell therapies for cerebral ischemia.

Heads and Tails of Natriuretic Peptides: Neuroprotective Role of Brain Natriuretic Peptide.

BACKGROUND: Besides the relevant role of brain-type natriuretic peptide (BNP) as biomarker of cardioembolic strokes, new experimental evidences suggest that this peptide may mediate neuroprotective effects. In this study, we have evaluated for the first time the clinical association between BNP (by means of proBNP) and good outcome in ischemic stroke patients, and analyzed the effect of blood BNP increase in an ischemic animal model. METHODS AND RESULTS: A retrospective study with 2 different cohorts (262 patients in cohort I and 610 in cohort II) from the same prospective stroke registry was performed. proBNP concentration was analyzed within the first 12 hours from stroke onset. The primary predictor variable was functional outcome evaluated by modified Rankin Scale at 3 months. For the experimental study, BNP pretreatment was tested in an ischemic animal model subjected to a transient occlusion of the cerebral artery, and the infarct volume and sensorimotor deficit were evaluated for 14 days. Cardioembolic strokes presented a positive correlation between proBNP concentration and modified Rankin Scale at 3 months; however, noncardioembolic strokes presented a negative correlation. In the logistic regression analysis, noncardioembolic strokes with concentrations of proBNP ≥340 pg/mL were associated with a good outcome. In line with these clinical findings, the experimental study revealed that those BNP pretreated animals presented a reduction on infarct volumes at 24 hours and functional recovery at days 7 and 14 compared with the control groups. CONCLUSIONS: These clinical and experimental evidences support the potential role of BNP as a protective factor against cerebral ischemia.

Inclusion criteria update for the rat intraluminal ischaemic model for preclinical studies.

Proper occlusion of the medial cerebral artery, as determined by laser Doppler monitoring, during cerebral ischaemia in rat models is an important inclusion criterion in experimental studies. However, successful occlusion of the artery does not always guarantee a reproducible infarct volume, which is crucial for validating the efficacy of new protective drugs. In a rat intraluminal ischaemic model, laser Doppler monitoring alone was compared with laser Doppler monitoring in combination with magnetic resonance angiography (MRA) and diffusion-weighted imaging (DWI). Twenty-eight animals showed successful occlusion and reperfusion determined with Doppler monitoring, with an infarct size at 24 h of 16.7±11.5% (determined as ischaemic damage with respect to the ipsilateral hemisphere volume). However, when arterial occlusion and infarct damage were analysed in these animals using MRA and DWI, respectively, 15 animals were excluded and only 13 animals were included, with an infarct size at 24 h of 21.6±6.1%, showing a variability in the infarct size significantly lower (P<0.05, F-test) than that obtained with Doppler monitoring alone. We also observed that blocking of the pterygopalatine artery (a maxillary artery that is usually occluded in the intraluminal ischaemic model) was not relevant for this model, at least in terms of infarct variability. These results show that laser Doppler monitoring is a necessary procedure, but not sufficient to guarantee a reproducible infarct volume, in a rat ischaemic model. Therefore, laser Doppler monitoring in combination with DWI and MRA represents a reliable inclusion protocol during ischaemic surgery for the analysis of new protective drugs.