Gene expression analyses reveal potential mechanism of inorganic arsenic-induced apoptosis in zebrafish.

Our previous study showed that sodium arsenite (200 mg/L) affected the nervous system and induced motor neuron development via the Sonic hedgehog pathway in zebrafish larvae. To gain more insight into the effects of arsenite on other signaling pathways, including apoptosis, we have performed quantitative polymerase chain reaction array-based gene expression analyses. The 96-well array plates contained primers for 84 genes representing 10 signaling pathways that regulate several biological functions, including apoptosis. We exposed eggs at 5 h postfertilization until the 72 h postfertilization larval stage to 200 mg/L sodium arsenite. In the Janus kinase/signal transducers and activators of transcription, nuclear factor κ-light-chain-enhancer of activated B cells, and Wingless/Int-1 signaling pathways, the expression of only one gene in each pathway was significantly altered. The expression of multiple genes was altered in the p53 and oxidative stress pathways. Sodium arsenite induced excessive apoptosis in the larvae. This compelled us to analyze specific genes in the p53 pathway, including cdkn1a, gadd45aa, and gadd45ba. Our data suggest that the p53 pathway is likely responsible for sodium arsenite-induced apoptosis. In addition, sodium arsenite significantly reduced global DNA methylation in the zebrafish larvae, which may indicate that epigenetic factors could be dysregulated after arsenic exposure. Together, these data elucidate potential mechanisms of arsenic toxicity that could improve understanding of arsenic's effects on human health.

Tauroursodeoxycholic acid (TUDCA) is neuroprotective in a chronic mouse model of Parkinson's disease.

OBJECTIVE: Parkinson's disease (PD) is a progressive motor disease of unknown etiology. Although neuroprotective ability of endogenous bile acid, tauroursodeoxycholic acid (TUDCA), shown in various diseases, including an acute model of PD,the potential therapeutic role of TUDCA in progressive models of PD that exhibit all aspects of PD has not been elucidated. In the present study, mice were assigned to one of four treatment groups: (1) Probenecid (PROB); (2) TUDCA, (3) MPTP + PROB (MPTPp); and (3) TUDCA + MPTPp. Methods: Markers for dopaminergic function, neuroinflammation, oxidative stress and autophagy were assessed using high performance liquid chromatography (HPLC), immunohistochemistry (IHC) and western blot (WB) methods. Locomotion was measured before and after treatments. Results: MPTPp decreased the expression of dopamine transporters (DAT) and tyrosine hydroxylase (TH), indicating dopaminergic damage, and induced microglial and astroglial activation as demonstrated by IHC analysis. MPTPp also decreased DA and its metabolites as demonstrated by HPLC analysis. Further, MPTPp-induced protein oxidation; increased LAMP-1 expression indicated autophagy and the promotion of alpha-synuclein (α-SYN) aggregation. Discussion: Pretreatment with TUDCA protected against dopaminergic neuronal damage, prevented the microglial and astroglial activation, as well as the DA and DOPAC reductions caused by MPTPp. TUDCA by itself did not produce any significant change, with data similar to the negative control group. Pretreatment with TUDCA prevented protein oxidation and autophagy, in addition to inhibiting α-SYN aggregation. Although TUDCA pretreatment did not significantly affect locomotion, only acute treatment effects were measured, indicating more extensive assessments may be necessary to reveal potential therapeutic effects on behavior. Together, these results suggest that autophagy may be involved in the progression of PD and that TUDCA may attenuate these effects. The efficacy of TUDCA as a novel therapy in patients with PD clearly warrants further study.

Antidepressant Actions of Ketamine: Potential Role of L-Type Calcium Channels.

Recently, the United States Food and Drug Administration approved esketamine, the S-enantiomer of ketamine, as a fast-acting therapeutic drug for treatment-resistant depression. Although ketamine is known as an N-methyl-d-aspartate (NMDA) receptor antagonist, the underlying mechanisms of how it elicits an antidepressant effect, specifically at subanesthetic doses, are not clear and remain an advancing field of research interest. On the other hand, high-dose (more than the anesthetic dose) ketamine-induced neurotoxicity in animal models has been reported. There has been progress in understanding the potential pathways involved in ketamine-induced antidepressant effects, some of which include NMDA-receptor antagonism, modulation of voltage-gated calcium channels, and brain-derived neurotrophic factor (BDNF) signaling. Often these pathways have been shown to be linked. Voltage-gated L-type calcium channels have been shown to mediate the rapid-acting antidepressant effects of ketamine, especially involving induction of BDNF synthesis downstream, while BDNF deficiency decreases the expression of L-type calcium channels. This review focuses on the reported studies linking ketamine's rapid-acting antidepressant actions to L-type calcium channels with an objective to present a perspective on the importance of the modulation of intracellular calcium in mediating the effects of subanesthetic (antidepressant) versus high-dose ketamine (anesthetic and potential neurotoxicant), the latter having the ability to reduce intracellular calcium by blocking the calcium-permeable NMDA receptors, which is implicated in potential neurotoxicity.

Nifedipine toxicity is exacerbated by acetyl l-carnitine but alleviated by low-dose ketamine in zebrafish in vivo.

Calcium channel blocker (CCB) poisoning is a common and sometimes life-threatening emergency. Our previous studies have shown that acetyl l-carnitine (ALCAR) prevents cardiotoxicity and developmental toxicity induced by verapamil, a CCB used to treat patients with hypertension. Here, we tested whether toxicities of nifedipine, a dihydropyridine CCB used to treat hypertension, can also be mitigated by co-treatment with ALCAR. In the zebrafish embryos at three different developmental stages, nifedipine induced developmental toxicity with pericardial sac edema in a dose-dependent manner, which were surprisingly exacerbated with ALCAR co-treatment. Even with low-dose nifedipine (5 µm), when the pericardial sac looked normal, ALCAR co-treatment showed pericardial sac edema. We hypothesized that toxicity by nifedipine, a vasodilator, may be prevented by ketamine, a known vasoconstrictor. Nifedipine toxicity in the embryos was effectively prevented by co-treatment with low (subanesthetic) doses (25-100 µm added to the water) of ketamine, although a high dose of ketamine (2 mm added to the water) partially prevented the toxicity.As expected of a CCB, nifedipine either in the presence or absence of ketamine-reduced metabolic reactive oxygen species (ROS), a downstream product of calcium signaling, in the rapidly developing digestive system. However, nifedipine induced ROS in the trunk region that showed significantly stunted growth indicating that the tissues under stress potentially produced pathologic ROS. To the best of our knowledge, these studies for the first time show that nifedipine and the dietary supplement ALCAR together induce adverse effects while providing evidence on the therapeutic efficacy of subanesthetic doses of ketamine against nifedipine toxicity in vivo.

Acetyl L-carnitine targets adenosine triphosphate synthase in protecting zebrafish embryos from toxicities induced by verapamil and ketamine: An in vivo assessment.

Verapamil is a Ca2+ channel blocker and is highly prescribed as an anti-anginal, antiarrhythmic and antihypertensive drug. Ketamine, an antagonist of the Ca2+ -permeable N-methyl-d-aspartate-type glutamate receptors, is a pediatric anesthetic. Previously we have shown that acetyl l-carnitine (ALCAR) reverses ketamine-induced attenuation of heart rate and neurotoxicity in zebrafish embryos. Here, we used 48 h post-fertilization zebrafish embryos that were exposed to relevant drugs for 2 or 4 h. Heart beat and overall development were monitored in vivo. In 48 h post-fertilization embryos, 2 mm ketamine reduced heart rate in a 2 or 4 h exposure and 0.5 mm ALCAR neutralized this effect. ALCAR could reverse ketamine's effect, possibly through a compensatory mechanism involving extracellular Ca2+ entry through L-type Ca2+ channels that ALCAR is known to activate. Hence, we used verapamil to block the L-type Ca2+ channels. Verapamil was more potent in attenuating heart rate and inducing morphological defects in the embryos compared to ketamine at specific times of exposure. ALCAR reversed cardiotoxicity and developmental toxicity in the embryos exposed to verapamil or verapamil plus ketamine, even in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, an inhibitor of intracellular Ca2+ release suggesting that ALCAR acts via effectors downstream of Ca2+ . In fact, ALCAR's protective effect was blunted by oligomycin A, an inhibitor of adenosine triphosphate synthase that acts downstream of Ca2+ during adenosine triphosphate generation. We have identified, for the first time, using in vivo studies, a downstream effector of ALCAR that is critical in abrogating ketamine- and verapamil-induced developmental toxicities. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Cyclosporine exacerbates ketamine toxicity in zebrafish: Mechanistic studies on drug-drug interaction.

Cyclosporine A (CsA) is an immunosuppressive drug commonly used in organ transplant patients to prevent allograft rejections. Ketamine is a pediatric anesthetic that noncompetitively inhibits the calcium-permeable N-methyl-d-aspartic acid receptors. Adverse drug-drug interaction effects between ketamine and CsA have been reported in mammals and humans. However, the mechanism of such drug-drug interaction is unclear. We have previously reported adverse effects of combination drugs, such as verapamil/ketamine and shown the mechanism through intervention by other drugs in zebrafish embryos. Here, we show that ketamine and CsA in combination produce developmental toxicity even leading to lethality in zebrafish larvae when exposure began at 24 h post-fertilization (hpf), whereas CsA did not cause any toxicity on its own. We also demonstrate that acetyl l-carnitine (ALCAR) completely reversed the adverse effects. Both ketamine and CsA are CYP3A4 substrates. Although ketamine and CsA independently altered the expression of the hepatic marker CYP3A65, a zebrafish ortholog of human CYP3A4, both drugs together induced further increase in CYP3A65 expression. In the presence of ALCAR, however, CYP3A65 expression was normalized. ALCAR has been shown to prevent ketamine toxicity in mammal and zebrafish. In conclusion, CsA exacerbated ketamine toxicity and ALCAR reversed the effects. These results, providing evidence for the first time on the reversal of the adverse effects of CsA/ketamine interaction by ALCAR, would prove useful in addressing potential occurrences of such toxicities in humans. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Post-treatment with an ultra-low dose of NADPH oxidase inhibitor diphenyleneiodonium attenuates disease progression in multiple Parkinson's disease models.

Nicotinamide adenine dinucleotide phosphate oxidase, a key superoxide-producing enzyme, plays a critical role in microglia-mediated chronic neuroinflammation and subsequent progressive dopaminergic neurodegeneration in Parkinson's disease. Although nicotinamide adenine dinucleotide phosphate oxidase-targeting anti-inflammatory therapy for Parkinson's disease has been proposed, its application in translational research remains limited. The aim of this study was to obtain preclinical evidence supporting this therapeutic strategy by testing the efficacy of an ultra-low dose of the nicotinamide adenine dinucleotide phosphate oxidase inhibitor diphenyleneiodonium in both endotoxin (lipopolysaccharide)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using post-treatment regimens. Our data revealed that post-treatment with diphenyleneiodonium significantly attenuated progressive dopaminergic degeneration and improved rotarod activity. Remarkably, post-treatment with diphenyleneiodonium 10 months after lipopolysaccharide injection when mice had 30% loss of nigral dopaminergic neurons, showed high efficacy in protecting the remaining neuronal population and restoring motor function. Diphenyleneiodonium-elicited neuroprotection was associated with the inhibition of microglial activation, a reduction in the expression of proinflammatory factors and an attenuation of α-synuclein aggregation. A pathophysiological evaluation of diphenyleneiodonium-treated mice, including assessment of body weight, organs health, and neuronal counts, revealed no overt signs of toxicity. In summary, infusion of ultra-low dose diphenyleneiodonium potently reduced microglia-mediated chronic neuroinflammation by selectively inhibiting nicotinamide adenine dinucleotide phosphate oxidase and halted the progression of neurodegeneration in mouse models of Parkinson's disease. The robust neuroprotective effects and lack of apparent toxic side effects suggest that diphenyleneiodonium at ultra-low dose may be a promising candidate for future clinical trials in Parkinson's disease patients.

Substance P exacerbates dopaminergic neurodegeneration through neurokinin-1 receptor-independent activation of microglial NADPH oxidase.

Although dysregulated substance P (SP) has been implicated in the pathophysiology of Parkinson's disease (PD), how SP affects the survival of dopaminergic neurons remains unclear. Here, we found that mice lacking endogenous SP (TAC1(-/-)), but not those deficient in the SP receptor (neurokinin-1 receptor, NK1R), were more resistant to lipopolysaccharide (LPS)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic neurodegeneration than wild-type controls, suggesting a NK1R-independent toxic action of SP. In vitro dose-response studies revealed that exogenous SP enhanced LPS- and 1-methyl-4-phenylpyridinium (MPP(+))-induced dopaminergic neurodegeneration in a bimodal manner, peaking at submicromolar and subpicomolar concentrations, but was substantially less effective at intermediate concentrations. Mechanistically, the actions of submicromolar levels of SP were NK1R-dependent, whereas subpicomolar SP-elicited actions required microglial NADPH oxidase (NOX2), the key superoxide-producing enzyme, but not NK1R. Subpicomolar concentrations of SP activated NOX2 by binding to the catalytic subunit gp91(phox) and inducing membrane translocation of the cytosolic subunits p47(phox) and p67(phox). The importance of NOX2 was further corroborated by showing that inhibition or disruption of NOX2 blocked subpicomolar SP-exacerbated neurotoxicity. Together, our findings revealed a critical role of microglial NOX2 in mediating the neuroinflammatory and dopaminergic neurodegenerative effects of SP, which may provide new insights into the pathogenesis of PD.

Porcine brain microvessel endothelial cells show pro-inflammatory response to the size and composition of metallic nanoparticles.

The purpose of the current studies was to determine if systemic exposure of various metallic nanoparticles differing in size and composition [silver (Ag-NPs, 25, 40 and 80 nm), copper-oxide (Cu-NPs, 40 and 60 nm) or gold (Au-NPs, 3 and 5 nm)] can induce the release of pro-inflammatory mediators that influence the restrictive nature of the blood-brain barrier (BBB) in vitro. Confluent porcine brain microvessel endothelial cells (pBMECs) (8-12 days) were treated with various metallic nanoparticles (15 µg/ml). Extracellular concentrations of pro-inflammatory mediators (IL-1ß, TNFα and PGE2) were evaluated using ELISA. pBMECs were cultured in standard 12-well Transwell® inserts, and permeability was evaluated by measuring the transport of fluorescein across the pBMEC monolayers. PGE2 release following Cu-NP exposure was significantly increased when compared to the control. Similar results were observed for Ag-NPs but not Au-NPs. The secretion of TNFα and IL-1ß was observed for both Cu-NPs and Ag-NPs but not in response to Au-NPs. The post-treatment time profiles of TNFα and IL-1ß revealed that the IL-1ß response was more persistent. The permeability ratios (exposure/control) were significantly greater following exposure to Cu-NPs or Ag-NPs, compared to Au-NPs. Together, these data suggest that the composition and size of NPs can cause significant pro-inflammatory response that can influence the integrity of the BBB.

Inorganic arsenic alters the development of dopaminergic neurons but not serotonergic neurons and induces motor neuron development via Sonic hedgehog pathway in zebrafish.

The mechanism of inorganic arsenic-induced neurotoxicity at the cellular level is not known. In zebrafish, teratological effects of inorganic arsenic have been shown at various concentrations. Here, we used similar concentrations of inorganic arsenic to evaluate the effects on specific neuron types. Exposure of zebrafish embryos at 5 h post fertilization (hpf) to sodium arsenite induced developmental toxicity (reduced body length) in 72 hpf larvae, beginning at a concentration of 300 mg/L concentration. Mortality or overt morphological deformity was detected at 500 mg/L sodium arsenite. While 200 mg/L sodium arsenite induced development of tyrosine hydroxylase-positive (dopaminergic) neurons, there was no significant effect on the development of 5-hydroxytryptamine (serotonergic) neurons. Sodium arsenite reduced acetylcholinesterase activity. In the hb9-GFP transgenic larvae, both 200 and 400 mg/L sodium arsenite produced supernumerary motor neurons in the spinal cord. Inhibition of the Sonic hedgehog (Shh) pathway that is essential for motor neuron development, by Gant61, prevented sodium arsenite-induced supernumerary motor neuron development. Inductively coupled plasma mass spectrometry (ICP-MS) revealed that with 200 mg/L and 400 mg/L sodium arsenite treatment, each larva had an average of 387.8 pg and 847.5 pg arsenic, respectively. The data show for the first time that inorganic arsenic alters the development of dopaminergic and motor neurons in the zebrafish larvae and the latter occurs through the Shh pathway. These results may help understand why arsenic-exposed populations suffer from psychiatric disorders and motor neuron disease and Shh may, potentially, serve as a plasma biomarker of arsenic toxicity.

N-acetylcysteine prevents verapamil-induced cardiotoxicity with no effect on the noradrenergic arch-associated neurons in zebrafish.

There is a strong association between calcium channel blockers (CCBs) and heart failure. CCB toxicity is very common due to overdose and underlying medical conditions. CCBs also have been shown to affect the nervous system. Recently, we demonstrated that the antioxidant N-acetylcysteine (NAC) prevented ketamine-induced cardiotoxicity, developmental toxicity and neurotoxicity. Functionally, we attributed NAC's beneficial effect to its ability to increase cellular calcium. Here, we hypothesized that if there was an involvement of calcium in NAC's preventative effects on ketamine toxicity, NAC might also ameliorate toxicities induced by verapamil, an L-type CCB used to treat hypertension. Using zebrafish embryos, we show that in the absence of NAC, verapamil (up to 100 µM) dose-dependently reduced heart rate and those effects were prevented by NAC co-treatment. Furthermore, a 2-h treatment with NAC rescued reduction of heart rate induced by pre-treatment of 50 and 100 µM of verapamil for 18 h. Verapamil up to 100 µM and NAC up to 1.5 mM did not have any adverse effects on the expression of tyrosine hydroxylase in the noradrenergic neurons of the arch-associated cluster (AAC) located near the heart. NAC did not change cysteine levels in the embryos suggesting that the beneficial effect of NAC on verapamil toxicity may not involve its antioxidant property. In our search for compounds that can prevent CCB toxicity, this study, for the first time, demonstrates protective effects of NAC against verapamil's adverse effects on the heart.

Impaired Amyloid Beta Clearance and Brain Microvascular Dysfunction are Present in the Tg-SwDI Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) pathology is characterized by amyloid plaques containing amyloid beta (Aß) peptides, neurofibrillary tangles containing hyperphosphorylated tau protein, and neuronal loss. In addition, Aß deposition in brain microvessels, known as cerebral amyloid angiopathy (CAA), increases blood-brain barrier (BBB) permeability and induces vascular dysfunction which aggravates AD pathology. The aim of the present study was to characterize neurovascular dysfunction in the Tg-SwDI mouse model of AD. Isolated brain capillaries from wild type (WT) and Tg-SwDI mice were used to evaluate the expression of monomeric and aggregated forms of Aß, P-glycoprotein (P-gp), the receptor for advance glycation end-products (RAGE) and the tight junction (TJs) proteins occludin and claudin-5. Cultured brain endothelial cells were used to analyze barrier function via fluorescein flux. Isolated capillaries from Tg-SwDI mice contained increased levels of aggregated and oligomeric Aß compared to WT animals. Isolated capillaries from Tg-SwDI had decreased levels of P-gp, which transports Aß from brain to blood, and increased levels of RAGE, which transports Aß from blood to brain. In addition, the TJ protein occludin was decreased in Tg-SwDI mice relative to WT mice, which correlated with an increase in BBB permeability in cultured brain endothelial cells. These findings demonstrated that Tg-SwDI mice exhibit Aß aggregation that is due, in part, to impaired Aß clearance driven by both a decrease in P-gp and increase in RAGE protein levels in brain capillaries. Aß aggregation promotes a decrease in the expression of the TJ protein occludin, and as consequence an increase in BBB permeability.

Neuroprotective effects of acetyl-l-carnitine (ALC) in a chronic MPTP-induced Parkinson's disease mouse model: Endothelial and microglial effects.

Parkinson's disease (PD) is a progressive motor disease with clinical features emerging due to degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), which project to the caudate putamen (CPu) where they release dopamine (DA). The current study investigated whether acetyl-l-carnitine (ALC) could ameliorate the pathology seen in an in vivoin vivo chronic 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of PD. Four treatment groups were included: 1) CONTROL receiving probenecid (PROB; 250 mg/kg) only, 2) MPTP (25 mg/kg) + PROB, 3) MPTP + ALC (100 mg/kg), and 4) ALC alone. MPTP-induced losses in tyrosine hydroxylase and DA transporter immunoreactivity in the SNc and CPu were significantly reduced by ALC. HPLC data further suggests that decreases in CPu DA levels produced by MPTP were also attenuated by ALC. Additionally, microglial activation and astrocytic reactivity induced by MPTP were greatly reduced by ALC, indicating protection against neuroinflammation. Glucose transporter-1 and the tight junction proteins occludin and zonula occludins-1 were also protected from MPTP-induced down-regulation by ALC. Together, data suggest that in this model, ALC protects against MPTP-induced damage to endothelial cells and loss of DA neurons in the SNc and CPu, suggesting that ALC therapy may have the potential to slow or ameliorate the progression of PD pathology in a clinical setting.

Ketamine-induced attenuation of reactive oxygen species in zebrafish is prevented by acetyl l-carnitine in vivo.

Ketamine, an anesthetic, is a non-competitive antagonist of the calcium-permeable N-methyl-d-aspartate (NMDA) receptor. High concentrations of ketamine have been implicated in cardiotoxicity and neurotoxicity. Often, these toxicities are thought to be mediated by reactive oxygen species (ROS). However, findings to the contrary showing ketamine reducing ROS in mammalian cells and neurons in vitro, are emerging. Here, we determined the effects of ketamine on ROS levels in zebrafish larvae in vivo. Based on our earlier studies demonstrating reduction in ATP levels by ketamine, we hypothesized that as a calcium antagonist, ketamine would also prevent ROS generation, which is a by-product of ATP synthesis. To confirm that the detected ROS in a whole organism, such as the zebrafish larva, is specific, we used diphenyleneiodonium (DPI) that blocks ROS production by inhibiting the NADPH Oxidases (NOX). Upon 20 h exposure, DPI (5 and 10 µM) and ketamine at (1 and 2 mM) reduced ROS in the zebrafish larvae in vivo. Using acetyl l-carnitine (ALCAR), a dietary supplement, that induces mitochondrial ATP synthesis, we show elevated ROS generation with increasing ALCAR concentrations. Combined, ketamine and ALCAR counter-balanced ROS generation in the larvae suggesting that ketamine and ALCAR have opposing effects on mitochondrial metabolism, which may be key to maintaining ROS homeostasis in the larvae and affords ALCAR the ability to prevent ketamine toxicity. These results for the first time show ketamine's antioxidative and ALCAR's prooxidative effects in a live vertebrate.

Characterization of Serum Exosomes from a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's Disease (AD) is the most common type of dementia characterized by amyloid plaques containing Amyloid Beta (Aß) peptides and neurofibrillary tangles containing tau protein. In addition to neuronal loss, Cerebral Amyloid Angiopathy (CAA) commonly occurs in AD. CAA is characterized by Aß deposition in brain microvessels. Recent studies have suggested that exosomes (cell-derived vesicles containing a diverse cargo) may be involved in the pathogenesis of AD. OBJECTIVE: Isolate and characterize brain-derived exosomes from a transgenic mouse model of AD that presents CAA. METHODS: Exosomes were isolated from serum obtained from 13-month-old wild type and AD transgenic female mice using an exosome precipitation solution. Characterization of exosomal proteins was performed by western blots and dot blots. RESULTS: Serum exosomes were increased in transgenic mice compared to wild types as determined by increased levels of the exosome markers flotillin and alix. High levels of neuronal markers were found in exosomes, without any difference any between the 2 groups. Markers for endothelial-derived exosomes were decreased in the transgenic model, while astrocytic-derived exosomes were increased. Exosome characterization showed increased levels of oligomeric Aß and oligomeric and monomeric forms tau on the transgenic animals. Levels of amyloid precursor protein were also increased. In addition, pathological and phosphorylated forms of tau were detected, but no difference was observed between the groups. CONCLUSION: These data suggest that monomeric and oligomeric forms of Aß and tau are secreted into serum via brain exosomes, most likely derived from astrocytes in the transgenic mouse model of AD with CAA. Studies on the implication of this event in the propagation of AD are underway.

N-acetylcysteine prevents ketamine-induced adverse effects on development, heart rate and monoaminergic neurons in zebrafish.

N-acetylcysteine, a precursor molecule of glutathione, is an antioxidant. Ketamine, a pediatric anesthetic, has been implicated in cardiotoxicity and neurotoxicity including modulation of monoaminergic systems in mammals and zebrafish. Here, we show that N-acetylcysteine prevents ketamine's adverse effects on development and monoaminergic neurons in zebrafish embryos. The effects of ketamine and N-acetylcysteine alone or in combination were measured on the heart rate, body length, brain serotonergic neurons and tyrosine hydroxylase-immunoreactive (TH-IR) neurons. In the absence of N-acetylcysteine, a concentration of ketamine that produces an internal embryo exposure level comparable to human anesthetic plasma concentrations significantly reduced heart rate and body length and those effects were prevented by N-acetylcysteine co-treatment. Ketamine also reduced the areas occupied by serotonergic neurons in the brain, whereas N-acetylcysteine co-exposure counteracted this effect. TH-IR neurons in the embryo brain and TH-IR cells in the trunk were significantly reduced with ketamine treatment, but not in the presence of N-acetylcysteine. In our continued search for compounds that can prevent ketamine toxicity, this study using specific endpoints of developmental toxicity, cardiotoxicity and neurotoxicity, demonstrates protective effects of N-acetylcysteine against ketamine's adverse effects. This is the first study that shows the protective effects of N-acetylcysteine on ketamine-induced developmental defects of monoaminergic neurons as observed in a whole organism.

Identification of altered microRNAs in serum of a mouse model of Parkinson's disease.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, whose hallmark is the loss of dopamine terminals in the substantia nigra pars compacta (SNpc). PD is usually diagnosed after the appearance of motor symptoms, when about 70% of neurons in the SNpc have already been lost. Because of that, it is important to search for new methods that aid in the early diagnosis of PD. In recent years, microRNAs (miRs) have emerged as potential biomarkers for a variety of diseases and hold the potential to be used to aid in the diagnosis of PD. Therefore, the aim of this study was to characterize if specific miRs are differentially expressed in serum in a mouse model of PD. To induce PD-like damage, mice were subcutaneously injected with 25 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) by administering 10 doses over a period of 5 weeks, with 3.5 days between doses. Expression of 71 different microRNAs was quantified in serum separated from blood collected at day 35, using next-generation sequencing. Histological analysis and quantification of neurotransmitters were performed to confirm dopaminergic neurodegeneration. Chronic MPTP treatment induced loss of dopaminergic terminals in the SNpc and caudate putamen, confirmed by a decrease in the number of tyrosine hydroxylase and dopamine transporter positive cells. In addition, MPTP decreased the concentration of dopamine and its metabolites in the SNpc, simulating the damage observed in PD. From the 71 miRs analyzed, only 4 were differentially expressed after MPTP treatment. Serum levels of miR19b, miR124, miR126a and miR133b were significantly decreased in MPTP-treated mice compared to control. These data suggest that specific miRs are downregulated in a pre-clinical model of PD and hold the potential to be used as biomarkers to aid in the diagnosis of this disease. Further experiments need to be conducted to validate the use of these miRs as biomarkers of PD in additional pre-clinical models as well as in samples from patients diagnosed with PD.

Mechanistic studies on ketamine-induced mitochondrial toxicity in zebrafish embryos.

Ketamine, a phencyclidine derivative, is an antagonist of the Ca2+-permeable N-methyl-d-aspartate (NMDA)-type glutamate receptors. It is a pediatric anesthetic and has been implicated in developmental neurotoxicity. Ketamine has also been shown to deplete ATP in mammalian cells. Our previous studies showed that acetyl l-carnitine (ALCAR) prevented ketamine-induced cardiotoxicity and neurotoxicity in zebrafish embryos. Based on our finding that ALCAR's protective effect was blunted by oligomycin A, an inhibitor of ATP synthase, we further investigated the effects of ketamine and ALCAR on ATP levels, mitochondria and ATP synthase in zebrafish embryos. The results demonstrated that ketamine reduced ATP levels in the embryos but not in the presence of ALCAR. Ketamine reduced total mitochondrial protein levels and mitochondrial potential, which were prevented with ALCAR co-treatment. To determine the cause of ketamine-induced ATP deficiency, we explored the status of ATP synthase. The results showed that a subunit of ATP synthase, atp5α1, was transcriptionally down-regulated by ketamine, but not in the presence of ALCAR, although ketamine caused a significant upregulation in another ATP synthase subunit, atp5ß and total ATP synthase protein levels. Most of the ATP generated by heart mitochondria are utilized for its contraction and relaxation. Ketamine-treated embryos showed abnormal heart structure, which was abolished with ALCAR co-treatment. This study offers evidence for a potential mechanism by which ketamine could cause ATP deficiency mediated by mitochondrial dysfunction.

The time course of blood brain barrier leakage and its implications on the progression of methamphetamine-induced seizures.

The initial goals of these experiments were to determine: 1) if blood-brain barrier (BBB) breakdown was a cause or an effect of METH-induced seizures; 2) all the brain regions where BBB is disrupted as seizures progress; and 3) the correlations between body temperature and vascular leakage and neurodegeneration. A fourth objective was added after initial experimentation to determine if sub-strain differences existed in adult male C57 B6 J (Jackson laboratories, JAX) versus C57 B6N (Charles River, CR) mice involving their susceptibility to BBB breakdown and seizure severity. With the 1st "maximal" intensity myoclonic-tonic seizure (MCT) varying degrees of IgG infiltration across the BBB (≤1 mm2) were prominent in olfactory system (OS) associated regions and in thalamus, hypothalamus and neocortex. IgG infiltration areas in the OS-associated regions of the bed nucleus of the stria terminalis, septum and more medial amygdala nuclei, and the hypothalamus were increased significantly by the time continuous behavioral seizures (CBS) developed. Mice receiving METH that had body temperatures of ≥40 °C had IgG infiltration along with MCT or CBS but peak body temperatures above 40 °C did not significantly increase IgG infiltration. Neurodegeneration seen at ≥6 h was restricted to the OS in both JAX and CR mice and was most prominent in the posteromedial cortical amygdaloid nucleus. Neurodegeneration in the anterior septum (tenia tecta) was seen only in the JAX mice. We hypothesize that METH-induced hypertension and hyperthermia lead to BBB breakdown and other vascular dysfunctions in the OS brain regions resulting in OS hyperexcitation. Excitation of the OS neural network then leads to the development of seizures. These seizures in turn exacerbate the energy depletions and the reactive oxygen stress produced by hyperthermia further damaging the BBB and vascular function. These events form a recurrent cycle that results in ever increasing seizure activity and neurotoxicity.

Changes in the metabolome and microRNA levels in biological fluids might represent biomarkers of neurotoxicity: A trimethyltin study.

Neurotoxicity has been linked with exposure to a number of common drugs and chemicals, yet efficient, accurate, and minimally invasive methods to detect it are lacking. Fluid-based biomarkers such as those found in serum, plasma, urine, and cerebrospinal fluid have great potential due to the relative ease of sampling but at present, data on their expression and translation are lacking or inconsistent. In this pilot study using a trimethyl tin rat model of central nervous system toxicity, we have applied state-of-the-art assessment techniques to identify potential individual biomarkers and patterns of biomarkers in serum, plasma, urine or cerebral spinal fluid that may be indicative of nerve cell damage and degeneration. Overall changes in metabolites and microRNAs were observed in biological fluids that were associated with neurotoxic damage induced by trimethyl tin. Behavioral changes and magnetic resonance imaging T2 relaxation and ventricle volume changes served to identify animals that responded to the adverse effects of trimethyl tin. Impact statement These data will help design follow-on studies with other known neurotoxicants to be used to assess the broad applicability of the present findings. Together this approach represents an effort to begin to develop and qualify a set of translational biochemical markers of neurotoxicity that will be readily accessible in humans. Such biomarkers could prove invaluable for drug development research ranging from preclinical studies to clinical trials and may prove to assist with monitoring of the severity and life cycle of brain lesions.