L-DOPA ameliorates hippocampus-based mitochondria respiratory dysfunction caused by GCI/R injury.

Mitochondrial dysmorphology/dysfunction follow global cerebral ischemia-reperfusion (GCI/R) injury, leading to neuronal death. Our previous researches demonstrated that Levodopa (L-DOPA) improves learning and memory impairment in GCI/R rats by increasing synaptic plasticity of hippocampal neurons. This study investigates if L-DOPA, used in Parkinson's disease treatment, alleviates GCI/R-induced cell death by enhancing mitochondrial quality. Metabolomics and transcriptomic results showed that GCI/R damage affected the Tricarboxylic acid (TCA) cycle in the hippocampus. The results of this study show that L-DOPA stabilized mitochondrial membrane potential and ultrastructure in hippocampus of GCI/R rats, increased dopamine level in hippocampus, decreased succinic acid level, and stabilized Ca2+ level in CA1 subregion of hippocampus. As a precursor of dopamine, L-DOPA is presumed to improves mitochondrial function in hippocampus of GCI/R rats. However, dopamine cannot cross the blood-brain barrier, so L-DOPA is used in clinical therapy to supplement dopamine. In this investigation, OGD/R models were established in isolated mouse hippocampal neurons (HT22) and primary rat hippocampal neurons. Notably, dopamine exhibited a multifaceted impact, demonstrating inhibition of mitochondrial reactive oxygen species (mitoROS) production, stabilization of mitochondrial membrane potential and Ca2+ level, facilitation of TCA circulation, promotion of aerobic respiratory metabolism, and downregulation of succinic acid-related gene expression. Consistency between in vitro and in vivo results underscores dopamine's significant neuroprotective role in mitigating mitochondrial dysfunction following global cerebral hypoxia and ischemia injury. Supplement dopamine may represent a promising therapy to the cognitive impairment caused by GCI/R injury.

Methylene blue and its potential in the treatment of traumatic brain injury, brain ischemia, and Alzheimer's disease.

Traumatic brain injury (TBI) and brain ischemia/reperfusion cause neurodegenerative processes that can continue after the acute stage with the development of severe brain atrophy with dementia. In this case, the long-term neurodegeneration of the brain is similar to the neurodegeneration characteristic of Alzheimer's disease (AD) and is associated with the accumulation of beta amyloid and tau protein. In the pathogenesis of AD as well as in the pathogenesis of cerebral ischemia and TBI oxidative stress, progressive inflammation, glial activation, blood-brain barrier dysfunction, and excessive activation of autophagy are involved, which implies the presence of many targets that can be affected by neuroprotectors. That is, multivariate cascades of nerve tissue damage represent many potential targets for therapeutic interventions. One of such substances that can be used in multi-purpose therapeutic strategies is methylene blue (MB). This drug can have an antiapoptotic and anti-inflammatory effect, activate autophagy, inhibit the aggregation of proteins with an irregular shape, inhibit NO synthase, and bypass impaired electron transfer in the respiratory chain of mitochondria. MB is a well-described treatment for methemoglobinemia, malaria, and encephalopathy caused by ifosfamide. In recent years, this drug has attracted great interest as a potential treatment for a number of neurodegenerative disorders, including the effects of TBI, ischemia, and AD.

Interaction between mitophagy, cadmium and zinc.

Mitophagy is the selective degradation of mitochondria by autophagy. This process is considered to be one of the stages of mitochondrial quality control, as a result of which damaged depolarized mitochondria are eliminated, thus limiting the formation of reactive oxygen species and the release of apoptogenic factors. Selective degradation of mitochondria by autophagy is one of the main ways to protect cells from cadmium toxicity, which results in dysfunction of the mitochondrial electron transport chain, leading to electron leakage, production of reactive oxygen species and cells death. However, excessive autophagy can be dangerous for cells. Currently, the participation of cadmium ions in normal physiological processes has not been detected. Zn2+, unlike Cd2+, regulate the activity of a large number of functionally important proteins, including transcription factors, enzymes, and adapters. It has been shown that Zn2+ not only participate in autophagy, but are also crucial for basal or induced autophagy. It is likely that zinc drugs can be used to reduce the cadmium toxicity and in the regulation of mithophagy.

Neuroprotective effects of methylene blue in streptozotocin-induced model of Alzheimer's disease.

Methylene blue (MB) can be used as a multidirectional neuroprotector to stop the development of multiple cascades of neuron damage during neurodegenerative processes. This study assesses a protective effect of MB, using an experimental simulation of sporadic Alzheimer's disease by intracerebroventricular administration of streptozotocin (STZ) in rats. It was found that a STZ-induced impairment of memory can be partially mitigated with intravenous injections of MB after the administration of STZ. The treatment of animals with MB prevented the STZ-induced increase in the number and density of microglial and GFAP-positive cells in the brain cortex. In addition, it was shown that the expression of the LC3B protein, an indicator of autophagy, increases in the hippocampus of animals treated with STZ. In the hippocampus of animals treated with MB, an increase in the expression of the LC3B protein was prevented. Using the Griess reaction assay and immunocytochemical study was found that MB reduces lipopolysaccharide-induced NO-production and the expression of iNOS in cultured neurons. In conclusion, our data demonstrate that MB has neuroprotective and anti-inflammatory effects and is able to prevent autophagy. These effects have important therapeutic implications, so MB could potentially play a role in the treatment of neurodegenerative processes.

Antioxidant Thymoquinone and Its Potential in the Treatment of Neurological Diseases.

Oxidative stress is one of the main pathogenic factors of neuron damage in neurodegenerative processes; this makes it an important therapeutic target to which the action of neuroprotectors should be directed. One of these drugs is thymoquinone. According to modern data, this substance has a wide range of pharmacological activity, including neuroprotective, which was demonstrated in experimental modeling of various neurodegenerative diseases and pathological conditions of the brain. The neuroprotective effect of thymoquinone is largely due to its antioxidant ability. Currently available data show that thymoquinone is an effective means to reduce the negative consequences of acute and chronic forms of cerebral pathology, leading to the normalization of the content of antioxidant enzymes and preventing an increase in the level of lipid peroxidation products. Antioxidant properties make this substance a promising basis for the development of prototypes of therapeutic agents aimed at the treatment of a number of degenerative diseases of the central nervous system.

Extracellular Alkalosis Reduces the Neurotoxicity of Zinc Ions in Cultured Cerebellar Granule Neurons.

Zn2+ is known to be important for the normal brain functions. Disruption of zinc homeostasis and zinc-induced neurotoxicity has been shown to play a role in the development of neurodegenerative diseases. In this work, we investigated the effect of extracellular alkalosis on the zinc ions neurotoxicity in the cultured rat cerebellar granule neurons. Zinc chloride (0.03-0.06 mM, 24 h) added to the culture medium of rat cerebellar granule neurons caused the dose-dependent death of these cells. According to ultrastructural morphological features, the process of cell death could be attributed to necrosis, since it was accompanied by swelling of intracellular organelles and disruption of cell membranes against the background of relatively intact nuclear membranes. Neuronal death was associated with an increase in the level of intracellular free zinc. The toxic effect of zinc ions was significantly decreased when ionotropic glutamate NMDA-receptors were blocked by MK-801 or when the extracellular pH was increased from 7.3 to 7.8, due to a decrease in the zinc overload of the cytoplasm of these cells. The presented results demonstrate that NMDA channels are one of the Zn ion entry pathways in the cultured cerebellar granule neurons. Extracellular alkalosis reduces the zinc overload of the cytoplasm and, consequently, promotes the survival of neurons. Probably, zinc's neurotoxicity is inextricably linked with changes in the intracellular concentration of protons.

Effect of zinc and copper ions on cadmium-induced toxicity in rat cultured cortical neurons.

BACKGROUND: Cadmium is a highly toxic heavy metal that is capable of accumulating in the body and causing neurodegeneration. However, the effect of other trace elements on Cd2+ toxicity is currently poorly understood. The aim of this work was to study the effect of Zn2+ and Cu2+ ions on cadmium-induced death of neurons in the cerebral cortex. METHODS: The work was performed on rat cortical primary cultures. The MTT test was used to determine the cytotoxicity effects. Analysis of intracellular Ca2+ concentration was assessed by the Fluo-4 AM calcium indicator that exhibit an increase in fluorescence upon binding Ca2+. MitoSOX Red (mitochondrial superoxide indicator) was used to measuring mitochondrial ROS content in live cells. RESULTS: In this article, we show that the administration of CdCl2 (0.005-0.02 mM) for 48 h induced an increase in dose-dependent death rate of cultured cortical neurons. Mature neurons were more sensitive to the damaging effects of Cd2+ than immature ones. ZnCl2 (0.01-0.03 mM) significantly protected neurons from this toxic effect. In contrast to ZnCl2, CuCl2 (0.01 mM) increased cadmium neurotoxicity. Using Fluo-4 AM, measurements of intracellular calcium ions demonstrated that 24 h-exposure to Cd2+ induced intensive increase in Fluo-4 fluorescence in neurons, which was significantly reduced by zinc ions. CuCl2 increased the cadmium-induced Fluo-4 and MitoSOX Red fluorescence in neurons. The chelator of intracellular Ca2+ BAPTA significantly decreased Cd2+-induced intensive increase in Fluo-4 fluorescence in cells. CONCLUSION: The data obtained by us indicate that Zn2+ and Cu2+ can affect the neurotoxicity of cadmium in different directions: Zn2+ weaken the violation of intracellular calcium homeostasis caused by cadmium, preventing cell death, while Cu2+ potentiate the increase in the level of free intracellular calcium induced by cadmium and the development of mitochondrial dysfunction with an increase in the production of free radicals in differentiated cultured neurons of the cerebral cortex, which ultimately stimulates cytotoxicity.

The Delayed Neuroprotective Effect of Methylene Blue in Experimental Rat Brain Trauma.

After traumatic brain injury (TBI), an increase in dysfunction of the limbs contralateral to injury focus was observed. Using different behavioral tests, we found that a single intravenous injection of methylene blue (MB, 1 mg/kg) 30 min after the injury reduced the impairment of the motor functions of the limbs from 7 to 120 days after TBI. Administration of methylene blue 30 min after the injury and then monthly (six injections in total) was the most effective both in terms of preservation of limb function and duration of therapeutic action. This therapeutic effect was clearly manifested from the seventh day and continued until the end of the experiment-by the 180th day after TBI. MB is known to possess antioxidant properties; it has a protective effect against TBI by promoting autophagy and minimizing lesion volume in the first two weeks after TBI. Studies of the brains on the 180th day after TBI demonstrated that the monthly treatment of animals with MB statistically significantly prevented an increase in the density of microglial cells in the ipsilateral hemisphere and a decrease in the thickness of the corpus callosum in the contralateral hemisphere in comparison with untreated animals. However, on the 180th day after TBI, the magnetic resonance imaging scan of the animal brains did not show a significant reduction in the volume of the lesion in MB-treated animals. These findings are important for understanding the development of the long-term effects of TBI and expand the required therapeutic window for targeted neuroprotective interventions.

The single intravenous administration of methylene blue after traumatic brain injury diminishes neurological deficit, blood-brain barrier disruption and decrease in the expression of S100 protein in rats.

The protective effect of methylene blue (MB) was investigated on the model of focal one-sided traumatic brain injury (TBI) of the sensorimotor cortex region from 1 to 7 days after the injury. TBI caused a reliable disruption of the functions of the limbs contralateral to injury focus, an increase in the expression of S100 protein and blood-brain barrier (BBB) permeability in the ipsilateral hemisphere. The single intravenous injection of MB (1 mg/kg body weight) 30 min after TBI significantly reduced the limb function impairment as well as a TBI-induced increase in the expression of inflammatory marker S100 protein, and BBB permeability. When modeling inflammation in vitro, MB was found to protect cultured neurons from the toxic effects of lipopolysaccharide. In conclusion, the preservation of blood-brain barrier and a decrease in the expression of S100 protein may be an important mechanism by means of which MB improves neurological outcome. Our data demonstrate that MB can be a very promising pharmacological compound with neuroprotective properties for TBI treatment.

Role of zinc and copper ions in the pathogenetic mechanisms of traumatic brain injury and Alzheimer's disease.

The disruption of homeostasis of zinc (Zn2+) and copper (Cu2+) ions in the central nervous system is involved in the pathogenesis of many neurodegenerative diseases, such as amyotrophic lateral sclerosis, Wilson's, Creutzfeldt-Jakob, Parkinson's, and Alzheimer's diseases (AD), and traumatic brain injury (TBI). The last two pathological conditions of the brain are the most common; moreover, it is possible that TBI is a risk factor for the development of AD. Disruptions of Zn2+ and Cu2+ homeostasis play an important role in the mechanisms of pathogenesis of both TBI and AD. This review attempts to summarize and systematize the currently available research data on this issue. The neurocytotoxicity of Cu2+ and Zn2+, the synergism of the toxic effect of calcium and Zn2+ ions on the mitochondria of neurons, and the interaction of Zn2+ and Cu2+ with ß-amyloid (Abeta) and tau protein are considered.

Mitochondria-Targeted Antioxidants as Potential Therapy for the Treatment of Traumatic Brain Injury.

The aim of this article is to review the publications describing the use of mitochondria-targeted antioxidant therapy after traumatic brain injury (TBI). Recent works demonstrated that mitochondria-targeted antioxidants are very effective in reducing the negative effects associated with the development of secondary damage caused by TBI. Using various animal models of TBI, mitochondria-targeted antioxidants were shown to prevent cardiolipin oxidation in the brain and neuronal death, as well as to markedly reduce behavioral deficits and cortical lesion volume, brain water content, and DNA damage. In the future, not only a more detailed study of the mechanisms of action of various types of such antioxidants needs to be conducted, but also their therapeutic values and toxicological properties are to be determined. Moreover, the optimal therapeutic effect needs to be achieved in the shortest time possible from the onset of damage to the nervous tissue, since secondary brain damage in humans can develop for a long time, days and even months, depending on the severity of the damage.

The single intravenous administration of mitochondria-targeted antioxidant SkQR1 after traumatic brain injury attenuates neurological deficit in rats.

The protective effect of SkQR1, a mitochondria-targeted antioxidant, was investigated on the model of focal one-sided traumatic brain injury (TBI) of the sensorimotor cortex region from 1 to 7 days after the injury. TBI caused a reliable disruption of the functions of the limbs contralateral to injury focus. The intravenous single injection of SkQR1 (250 nmol/kg) but not C12R1 (a SkQR1 homologue devoid of the antioxidant group) 30 min after TBI reduced the impairment of the motor functions of the limbs. A statistically significant improvement in limb function in animals was shown using 3 different tests: limb-placing test, beam-walking test and grip strength test. A pronounced therapeutic effect appeared on the 1th day and lasted until the end of the experiment - the 7th day after TBI. Histopathological examination showed that in the group of animals that did not receive SkQR1 in the marginal layer of the lesion there was a marked increase in astroglial expression, infiltration with segmented neutrophils, and poor survivability of neurons compared with animals treated with SkQR1. The obtained results demonstrate that the single use of plastoquinone-containing mitochondria-targeted antioxidant SkQR1 at the early stages of development of traumatic brain damage can reduce TBI-related disruptions of limb functions, and that mechanisms of the brain damage after trauma are dependent on the production of mitochondrial reactive oxygen species.

Neurogenesis and brain aging.

Human aging affects the entire organism, but aging of the brain must undoubtedly be different from that of all other organs, as neurons are highly differentiated postmitotic cells, for the majority of which the lifespan in the postnatal period is equal to the lifespan of the entire organism. In this work, we examine the distinctive features of brain aging and neurogenesis during normal aging, pathological aging (Alzheimer's disease), and accelerated aging (Hutchinson-Gilford progeria syndrome and Werner syndrome).

The delayed protective effect of GK-2, а dipeptide mimetic of Nerve Growth Factor, in a model of rat traumatic brain injury.

The delayed protective effect of GK-2, a dipeptide mimetic of Nerve Growth Factor, was investigated on the model of focal one-sided traumatic brain injury (TBI) of the sensorimotor cortex region on the 180th day after the injury. TBI caused a reliably disruption of the functions of the limbs contralateral to injury focus. The intraperitoneal administration of GK-2 (1 mg/kg) from 1st to 4th and from 7th to 10th days after TBI reduced the impairment of the motor functions of the limbs. This therapeutic effect significant manifested itself from the 7th day and continued until the end of the experiment - on the 180th day after TBI. Morphological studies of the animal brains on the 180th day after TBI demonstrated a decrease in the number of neurons in the V layer of the cerebral cortex and a decrease in the thickness of the corpus callosum. The treatment of animals with GK-2 after TBI statistically significant prevented a decrease in the density of neurons in the ipsilateral hemisphere and a decrease in the thickness of the corpus callosum in the contralateral hemisphere in comparison with untreated animals. Additionally, we showed in vitro that GK-2 exhibits neuroprotective properties under oxidative stress in primary hippocampal cultures. Our results demonstrate that the use of GK-2 at the early stages of development of traumatic brain damage can prevent such delayed damage as neuronal and axonal degeneration as well as reduce TBI-related disruptions of brain functions.

Streptozotocin toxicity in vitro depends on maturity of neurons.

Streptozotocin (STZ) is a glucosamine-nitrosourea compound that is particularly toxic to the insulin-producing beta-cells of the pancreas in mammals; it is used for experimental simulation of sporadic Alzheimer's disease by means of intracerebroventricular administration in vivo. Here we show that the application of 3-4 mM STZ to primary culture for 48 h induces neuronal death in immature (2-3 days in vitro) cultures of rat cerebellar granule cells. Mature cultures (7-8 days in vitro) were poorly sensitive to this toxic treatment. Immature cultures demonstrated a high expression of the protein PSA-NCAM, the marker of immature neurons, and they were insensitive to the toxic effect of glutamate. In mature cultures, this protein was poorly expressed, whereas neurons showed a very high sensitivity to the toxic effect of glutamate. Measurements of the concentration of intracellular free calcium ions ([Ca2+]i) showed that the STZ-induced [Ca2+]i increase in young neurons was six times higher than that in mature neurons. Our results show that STZ is very toxic for immature neurons and probably it can significantly impair neurogenesis.

N-acetyl-l-cysteine and Mn2+ attenuate Cd2+-induced disturbance of the intracellular free calcium homeostasis in cultured cerebellar granule neurons.

Cadmium is a highly toxic heavy metal that is capable of accumulating in the body via direct exposure or through the alimentary and respiratory tract, leading to neurodegeneration. In this article, we show that the application of CdCl2 (0.001-0.005mM) for 48h induced high dose-dependent death rate of cultured cerebellar granule neurons (CGNs). Unlike Trolox or vitamin E, antioxidant N-acetyl-l-cysteine (NAC, 1mM) and Mn2+ (0.0025-0.005mM) significantly protected CGNs from this toxic effect. Using Fluo-4 AM, measurements of intracellular calcium ions demonstrated that 24h-exposure to Cd2+ induced intensive increase of Fluo-4 fluorescence in neurons accompanied by mitochondria swelling. These data imply that the cadmium-induced Ca2+ increase is an important element in the death of neurons due to toxic effect of cadmium and the mechanism of protective action of manganese and NAC is mediated by the prevention of increase in calcium levels.

Accelerated aging and aging process in the brain.

One of the approaches to the research of the problem of aging is the study of genetic pathologies leading to accelerated aging, such as the Hutchinson-Gilford progeria syndrome, Werner syndrome, and Down syndrome. Probably, this approach can be used in an attempt to understand the neuronal mechanisms underlying normal and pathological brain aging. The analysis of the current state of scientific knowledge about these pathologies shows that in the Hutchinson-Gilford progeria and Werner syndrome, the rate of brain aging is significantly lower than the rate of whole body aging, whereas in Down syndrome, the brain ages faster than other organs due to amyloid-beta accumulation and chronic oxidative stress in the brain tissue. The main point of a previously proposed hypothesis is that the aging of higher animals and humans is associated with an increased level of reactive oxygen species in mitochondria with age, which activates apoptosis, thus reducing the number of functioning cells.

Effects of copper on viability and functional properties of hippocampal neurons in vitro.

Copper (Cu2+) is an essential metal presented in the mammalian brain and released from synaptic vesicles following neuronal depolarization. However, the disturbance of Cu2+ homeostasis results in neurotoxicity. In our study we performed for the first time a combined functional investigation of cultured hippocampal neurons under Cu2+ exposure, its effect on spontaneous spike activity of hippocampal neuronal network cultured on multielectrode array (MEA), and development of long-term potentiation (LTP) in acute hippocampal slices in the presence of Cu2+. Application of 0.2mM CuCl2 for 24h reduced viability of cultured neurons to 40±6%, whereas 0.01mM CuCl2 did not influence significantly on the neuronal survival. However, exposure to the action of 0.01mM Cu2+ resulted in pronounced reduction of network spike activity and abolished LTP induced by high-frequency stimulation of Schaffer's collaterals in CA1 pyramidal neurons of hippocampal slices. Antioxidant Trolox, the hydrosoluble vitamin E analogue, prevented neurotoxic effect and alterations of network activity under Cu2+ exposure, but didn't change the impairment of LTP in Cu2+-exposured hippocampal slices. We hypothesized that spontaneous network neuronal activity probably is one of the potential targets of Cu2+-induced neurotoxicity, in which free radicals can be involved. At the same time, it may be suggested that Cu2+-induced alterations of long-lasting trace processes (like LTP) are not mediated by oxidative damage.

Streptozotocin causes neurotoxic effect in cultured cerebellar granule neurons.

Streptozotocin (STZ) is a glucosamine-nitrosourea compound used for experimental simulation of sporadic Alzheimer's disease at intracerebroventricular administration in vivo. The studies of STZ influence on neurons of central nervous system performed on the primary cultures are practically absent. We have shown the application of STZ (1-5mM) in primary culture for 48h induced strong dose-dependent death in cultured cerebellar granule neurons. This toxic effect was decreased by pyruvate, insulin partially. Using the indicator Fluo-4 AM for measurements of intracellular calcium ions and tetramethylrhodamine ethyl ester (TMRE) for detection of changes of mitochondrial membrane potential in live cells we have shown that 5 h-exposure to STZ induced intensive increase of Fluo-4 and decrease TMRE fluorescence in neurons. STZ exposure caused considerable ultrastructural alterations in granule neurons: chromatin clumping, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. Probably, STZ significantly impaired glucose metabolism and mitochondrial function that, in turn, resulted in mitochondrial membrane potential damage, excessive calcium overload and neuronal death.

Neuroprotective properties of mitochondria-targeted antioxidants of the SkQ-type.

In 2008, using a model of compression brain ischemia, we presented the first evidence that mitochondria-targeted antioxidants of the SkQ family, i.e. SkQR1 [10-(6'-plastoquinonyl)decylrhodamine], have a neuroprotective action. It was shown that intraperitoneal injections of SkQR1 (0.5-1 µmol/kg) 1 day before ischemia significantly decreased the damaged brain area. Later, we studied in more detail the anti-ischemic action of this antioxidant in a model of experimental focal ischemia provoked by unilateral intravascular occlusion of the middle cerebral artery. The neuroprotective action of SkQ family compounds (SkQR1, SkQ1, SkQTR1, SkQT1) was manifested through the decrease in trauma-induced neurological deficit in animals and prevention of amyloid-ß-induced impairment of long-term potentiation in rat hippocampal slices. At present, most neurophysiologists suppose that long-term potentiation underlies cellular mechanisms of memory and learning. They consider inhibition of this process by amyloid-ß1-42 as an in vitro model of memory disturbance in Alzheimer's disease. Further development of the above studies revealed that mitochondria-targeted antioxidants could retard accumulation of hyperphosphorylated τ-protein, as well as amyloid-ß1-42, and its precursor APP in the brain, which are involved in developing neurodegenerative processes in Alzheimer's disease.