The Rescue on Reperfusion Damage in Cerebral Infarction by Nelonemdaz (RODIN) Trial: Protocol for a Double-Blinded Clinical Trial of Nelonemdaz in Patients with Hyperacute Ischemic Stroke and Endovascular Thrombectomy.

BACKGROUND AND PURPOSE: Nelonemdaz (Neu2000) has both selective antagonism against 2B subunit of N-methyl-D-aspartate receptor and antioxidant activity. This drug provides sufficient evidence of neuroprotection in acute cerebral ischemia/reperfusion models. This phase III trial aims to determine this effect in patients. DESIGN: The Rescue on Reperfusion Damage in Cerebral Infarction by Nelonemdaz is a multicenter, double-blinded clinical trial. A total of 496 patients will be randomly assigned into the nelonemdaz (a total of 5,250 mg divided by 10 times for 5 days) and placebo groups. Patients will be included if they have an acute ischemic stroke (National Institutes of Health Stroke Scale score ≥8) caused by intracranial large vessel occlusion in the anterior circulation (Alberta Stroke Program Early CT Score ≥4), and if they are expected to undergo endovascular thrombectomy within 12 hours after stroke onset. ENDPOINTS: The primary endpoint is a favorable shift in the modified Rankin Scale (mRS) score at 90 days after the first dose of drug. The data will be analyzed by the Cochran-Mantel-Haenszel shift test. The secondary endpoints include functional independence (mRS 0-2) at 35 and 90 days, the favorable shift of mRS at 35 days, the proportion of mRS 0 at 35 and 90 days, and the occurrence rates of symptomatic intracranial hemorrhage within 7 days. CONCLUSION: This trial will clarify the efficacy and safety of nelonemdaz in patients with acute ischemic stroke and endovascular thrombectomy. This study has been registered at ClinicalTrials. gov (NCT05041010).

Potent Analgesic Action of 2-acetoxy-5-(2-4 (trifluoromethyl)-phenethylamino)-benzoic Acid (Flusalazine) in Experimental Mice.

Purpose: Nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2 selective inhibitors are the most widely used drugs to treat pain. Conventional NSAIDs and COX-2 selective inhibitors, however, cause several side effects such as gastric damage, kidney damage, and cardiovascular problems. Our previous study showed that 2-acetoxy-5-(2-4-(trifluoromethyl)-phenethylamino)-benzoic acid ie, flusalazine (also known as ND-07), which exerts dual actions by serving both as an anti-inflammatory agent and a free radical scavenger, is an effective and safe treatment for severe inflammatory diseases in mice. The goal of the present study was to examine the potential analgesic action and safety of flusalazine in mice models of pain. Methods and Results: Flusalazine showed a significant analgesic effect in an acetic acid-induced abdominal constriction model. Likewise, total paw licking was reduced significantly in neurogenic (early stage) and inflammatory (late stage) pain induced by formalin in flusalazine-treated mice. In the tail immersion test, flusalazine significantly increased tail withdrawal time at 2 h after its administration. Also, the formation of paw edema in the flusalazine-treated group was significantly inhibited in a carrageenan-induced inflammatory pain model. Gastric damage was not induced by flusalazine even up to 1000 mg/kg, while aspirin and indomethacin caused critical gastric bleeding. Conclusion: These findings suggest that flusalazine's safety profile and analgesic effects have high translational potential for the clinical treatment of patients experiencing pain.

Nelonemdaz for Patients With Acute Ischemic Stroke Undergoing Endovascular Reperfusion Therapy: A Randomized Phase II Trial.

BACKGROUND: Nelonemdaz is a multitarget neuroprotectant that selectively blocks N-methyl-D-aspartate receptors and scavenges free radicals, as proven in preclinical ischemia-reperfusion studies. We aimed to evaluate the safety and efficacy of nelonemdaz in patients with acute ischemic stroke receiving endovascular reperfusion therapy. METHODS: This phase II randomized trial involved participants with large-artery occlusion in the anterior circulation at baseline who received endovascular reperfusion therapy <8 hours from symptom onset at 7 referral stroke centers in South Korea between October 29, 2016, and June 1, 2020. Two hundred thirteen patients were screened and 209 patients were randomly assigned at a 1:1:1 ratio using a computer-generated randomization system. Patients were divided into 3 groups based on the medication received-placebo, low-dose (2750 mg) nelonemdaz, and high-dose (5250 mg) nelonemdaz. The primary outcome was the proportion of patients with modified Rankin Scale scores of 0-2 at 12 weeks. RESULTS: Two hundred eight patients were assigned to the placebo (n=70), low-dose (n=71), and high-dose (n=67) groups. The groups had similar baseline characteristics. The primary outcome was achieved in 183 patients, and it did not differ among the groups (33/61 [54.1%], 40/65 [61.5%], and 36/57 [63.2%] patients; P=0.5578). The common odds ratio (90% CI) indicating a favorable shift in the modified Rankin Scale scores at 12 weeks was 1.55 (0.92-2.60) between the placebo and low-dose groups and 1.61 (0.94-2.76) between the placebo and high-dose groups. No serious adverse events were reported. CONCLUSIONS: The study arms showed no significant difference in the proportion of patients achieving modified Rankin Scale scores of 0-2 at 12 weeks. Nevertheless, nelonemdaz-treated patients showed a favorable tendency toward achieving these scores at 12 weeks, without serious adverse effects. Thus, a large-scale phase III trial is warranted. REGISTRATION: URL: https://clinicaltrials.gov; Unique identifier: NCT02831088.

Rationale and methods of the Antioxidant and NMDA receptor blocker Weans Anoxic brain damage of KorEa OHCA patients (AWAKE) trial.

BACKGROUND: Ischemic brain injury is a major hurdle that limits the survival of resuscitated out-of-hospital cardiac arrest (OHCA). METHODS: The aim of this study is to assess the feasibility and potential for reduction of ischemic brain injury in adult OHCA patients treated with high- or low-dose Neu2000K, a selective blocker of N-methyl-D-aspartate (NMDA) type 2B receptor and also a free radical scavenger, or given placebo. This study is a phase II, multicenter, randomized, double-blinded, prospective, intention-to-treat, placebo-controlled, three-armed, safety and efficacy clinical trial. This trial is a sponsor-initiated trial supported by GNT Pharma. Successfully resuscitated OHCA patients aged 19 to 80 years would be included. The primary outcome is blood neuron-specific enolase (NSE) level on the 3rd day. The secondary outcomes are safety, efficacy defined by study drug administration within 4 h in > 90% of participants, daily NSE up to 5th day, blood S100beta, brain MRI apparent diffusion coefficient imaging, cerebral performance category (CPC), and Modified Rankin Scale (mRS) at 5th, 14th, and 90th days. Assuming NSE of 42 ± 80 and 80 ± 80 µg/L in the treatment (high- and low-dose Neu2000K) and control arms with 80% power, a type 1 error rate of 5%, and a 28% of withdrawal prior to the endpoint, the required sample size is 150 patients. DISCUSSION: The AWAKE trial explores a new multi-target neuroprotectant for the treatment of resuscitated OHCA patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT03651557 . Registered on August 29, 2018.

Severe reactive astrocytes precipitate pathological hallmarks of Alzheimer's disease via H2O2- production.

Although the pathological contributions of reactive astrocytes have been implicated in Alzheimer's disease (AD), their in vivo functions remain elusive due to the lack of appropriate experimental models and precise molecular mechanisms. Here, we show the importance of astrocytic reactivity on the pathogenesis of AD using GiD, a newly developed animal model of reactive astrocytes, where the reactivity of astrocytes can be manipulated as mild (GiDm) or severe (GiDs). Mechanistically, excessive hydrogen peroxide (H2O2) originated from monoamine oxidase B in severe reactive astrocytes causes glial activation, tauopathy, neuronal death, brain atrophy, cognitive impairment and eventual death, which are significantly prevented by AAD-2004, a potent H2O2 scavenger. These H2O2--induced pathological features of AD in GiDs are consistently recapitulated in a three-dimensional culture AD model, virus-infected APP/PS1 mice and the brains of patients with AD. Our study identifies H2O2 from severe but not mild reactive astrocytes as a key determinant of neurodegeneration in AD.

Iron accumulation promotes TACE-mediated TNF-α secretion and neurodegeneration in a mouse model of ALS.

Oxidative stress contributes to degeneration of motor neurons in patients with amyotrophic lateral sclerosis (ALS) as well as transgenic mice overexpressing ALS-associated human superoxide dismutase 1 (SOD1) mutants. However, the molecular mechanism by which the ALS-linked SOD1 mutants including SOD1(G93A) induce oxidative stress remains unclear. Here, we show that iron was accumulated in ventral motor neurons from SOD1(G93A)-transgenic mice even at 4 weeks of age, subsequently inducing oxidative stress. Iron chelation with deferoxamine mesylate delayed disease onset and extended lifespan of SOD1(G93A) mice. Furthermore, SOD1(G93A)-induced iron accumulation mediated the increase in the enzymatic activity of TNF-α converting enzyme (TACE), leading to secretion of TNF-α at least in part through iron-dependent oxidative stress. Our findings suggest iron as a key determinant of early motor neuron degeneration as well as proinflammatory responses at symptomatic stage in SOD1(G93A) mice.

MST1 functions as a key modulator of neurodegeneration in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by loss of motor neurons. Dominant mutations in the gene for superoxide dismutase 1 (SOD1) give rise to familial ALS by an unknown mechanism. Here we show that genetic deficiency of mammalian sterile 20-like kinase 1 (MST1) delays disease onset and extends survival in mice expressing the ALS-associated G93A mutant of human SOD1. SOD1(G93A) induces dissociation of MST1 from a redox protein thioredoxin-1 and promotes MST1 activation in spinal cord neurons in a reactive oxygen species-dependent manner. Moreover, MST1 was found to mediate SOD1(G93A)-induced activation of p38 mitogen-activated protein kinase and caspases as well as impairment of autophagy in spinal cord motoneurons of SOD1(G93A) mice. Our findings implicate MST1 as a key determinant of neurodegeneration in ALS.

AAD-2004 Attenuates Progressive Neuronal Loss in the Brain of Tg-betaCTF99/B6 Mouse Model of Alzheimer Disease.

Alzheimer's disease (AD) is a neurodegenerative disease that proceeds with the age-dependent neuronal loss, an irreversible event which causes severe cognitive and psychiatric devastations. In the present study, we investigated whether the compound, AAD-2004 [2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobenzoic acid] which has anti-oxidant and anti-inflammatory properties, is beneficial for the brain of Tg-betaCTF99/B6 mice, a murine AD model that was recently developed to display age-dependent neuronal loss and neuritic atrophy in the brain. Administration of AAD-2004 in Tg-betaCTF99/B6 mice from 10 months to 18 months of age completely repressed the accumulation of lipid peroxidation in the brain. AAD-2004 markedly suppressed neuronal loss and neuritic atrophy, and partially reversed depleted expression of calbindin in the brain of Tg-beta-CTF99/B6. These results suggest that AAD-2004 affords neurodegeneration in the brain of AD mouse model.

Antioxidant properties of Neu2000 on mitochondrial free radicals and oxidative damage.

Neu2000 [2-hydroxy-5-(2,3,5,6-tetrafluoro-4 trifluoromethylbenzylamino) benzoic acid] is a dual-acting neuroprotective agent that functions both as a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist and a free radical scavenger. In the present study, we investigated the scavenging activity of Neu2000 on various classes of reactive oxygen species and reactive nitrogen species (ROS/RNS) as well as its efficacy for reducing free radicals and oxidative stress/damage induced in spinal cord mitochondrial preparations. Neu2000 exerted scavenging activity against superoxide, nitric oxide, and hydroxyl radicals, and efficiently scavenged peroxynitrite. In the mitochondrial studies, Neu2000 markedly inhibited ROS/RNS and hydrogen peroxide levels following antimycin treatment. In addition, Neu2000 effectively scavenged hydroxyl radicals generated by iron(III)-ascorbate, reduced protein carbonyl formation mediated by hydroxyl radicals and peroxynitrite, and prevented glutathione oxidation caused by tert-butyl hydroperoxide in isolated mitochondria. Interestingly, incubation of isolated mitochondria with Neu2000 followed by centrifugation and removal of the supernatant also resulted in a concentration-dependent decrease in lipid peroxidation. This observation suggests that Neu2000 enters mitochondria to target free radicals or indirectly affects mitochondrial function in a manner that promotes antioxidant activity. The results of the present study demonstrate that Neu2000 possesses potent in vitro antioxidant activity due, most likely, to its active phenoxy group.

Prevention effects of ND-07, a novel drug candidate with a potent antioxidative action and anti-inflammatory action, in animal models of severe acute pancreatitis.

Oxidative stress and inflammation both play major roles in the development of the acute pancreatitis. Currently, a pancreatic enzyme inhibitor with limited efficacy is only clinically available in a few countries, and antioxidants or non-steroidal anti-inflammatory drugs (NSAIDs) provide only partial tissue protection in acute pancreatitis animal models. Here, we introduce a new drug candidate for treating acute pancreatitis named ND-07 [chemical name: 2-acetoxy-5-(2-4-(trifluoromethyl)-phenethylamino)-benzoic acid] that exhibits both potent antioxidative and anti-inflammatory activities. In an electron spin resonance (ESR) study, ND-07 almost blocked hydroxyl radical generation as low as 0.05 µM and significantly suppressed DNA oxidation and cell death in a lipopolysaccharide (LPS)-stimulated pancreatic cell line. In a cerulein plus LPS-induced acute pancreatitis model, ND-07 pretreatment showed significant tissue protective effects, with reductions of serum amylase and lipase levels and pancreatic wet weights. ND-07 not only diminished the plasma levels of malondialdehyde (MDA) and nitric oxide but also significantly decreased prostaglandin E2 (PGE2) and expression of tumor necrotizing factor-alpha (TNF-α) in the pancreatic tissue. In a severe acute necrotizing pancreatitis model induced by a choline deficient, ethionine-supplemented (CDE) diet, ND-07 dramatically protected the mortality even without any death, providing attenuation of pancreas, lung, and liver damages as well as the reductions in serum levels of lactate dehydrogenase (LDH), amylase and lipase, MDA levels in the plasma and pancreatic tissues, plasma levels of TNF-α, and interleukin-1 (IL-1ß). These findings suggest that current dual synergistic action mechanisms of ND-07 might provide a superior protection for acute pancreatitis than conventional drug treatments.

Concurrent blockade of free radical and microsomal prostaglandin E synthase-1-mediated PGE2 production improves safety and efficacy in a mouse model of amyotrophic lateral sclerosis.

While free radicals and inflammation constitute major routes of neuronal injury occurring in amyotrophic lateral sclerosis (ALS), neither antioxidants nor non-steroidal anti-inflammatory drugs have shown significant efficacy in human clinical trials. We examined the possibility that concurrent blockade of free radicals and prostaglandin E(2) (PGE(2))-mediated inflammation might constitute a safe and effective therapeutic approach to ALS. We have developed 2-hydroxy-5-[2-(4-trifluoromethylphenyl)-ethylaminobenzoic acid] (AAD-2004) as a derivative of aspirin. AAD-2004 completely removed free radicals at 50 nM as a potent spin-trapping molecule and inhibited microsomal PGE(2) synthase-1 (mPGES-1) activity in response to both lipopolysaccharide-treated BV2 cell with IC(50) of 230 nM and recombinant human mPGES-1 protein with IC(50) of 249 nM in vitro. In superoxide dismutase 1(G93A) transgenic mouse model of ALS, AAD-2004 blocked free radical production, PGE(2) formation, and microglial activation in the spinal cords. As a consequence, AAD-2004 reduced autophagosome formation, axonopathy, and motor neuron degeneration, improving motor function and increasing life span. In these assays, AAD-2004 was superior to riluzole or ibuprofen. Gastric bleeding was not induced by AAD-2004 even at a dose 400-fold higher than that required to obtain maximal therapeutic efficacy in superoxide dismutase 1(G93A). Targeting both mPGES-1-mediated PGE(2) and free radicals may be a promising approach to reduce neurodegeneration in ALS and possibly other neurodegenerative diseases.

Role of the NMDA receptor and iron on free radical production and brain damage following transient middle cerebral artery occlusion.

Excess activation of ionotropic glutamate receptors and iron is believed to contribute to free radical production and neuronal death following hypoxic ischemia. We examined the possibility that both NMDA receptor activation and iron overload determine spatial and temporal patterns of free radical production after transient middle cerebral artery occlusion (tMCAO) in male Sprague-Dawley rats. Mitochondrial free radical (MFR) levels were maximally increased in neurons in the core at 1 h and 24 h after tMCAO. Early MFR production was blocked by administration of MK-801, an NMDA receptor antagonist, but not deferoxamine, an iron chelator. Neither MK-801 nor deferoxamine attenuated late MFR production in the core. Increased MFRs were observed in penumbral neurons within 6 h and gradually increased over 24 h after tMCAO. Slowly-evolving MFRs in the core and penumbra were accompanied by iron overload. Deferoxamine blocked iron overload but reduced MFR production only in the penumbra. Combined MK-801/deferoxamine reduced late MFR production in both core and penumbra in an additive manner. Combination therapy significantly ameliorated infarction compared with monotherapy. These findings suggest that the NMDA receptor activation and iron overload mediate late MFR production and infarction after tMCAO.

Effects of collagen-induced rheumatoid arthritis on amyloidosis and microvascular pathology in APP/PS1 mice.

BACKGROUND: Evidence suggests that rheumatoid arthritis (RA) may enhance or reduce the progression of Alzheimer's disease (AD). The present study was performed to directly explore the effects of collagen-induced rheumatoid arthritis (CIA) on amyloid plaque formation, microglial activation, and microvascular pathology in the cortex and hippocampus of the double transgenic APP/PS1 mouse model for AD. Wild-type or APP/PS1 mice that received type II collagen (CII) in complete Freund's adjuvant (CFA) at 2 months of age revealed characteristics of RA, such as joint swelling, synovitis, and cartilage and bone degradation 4 months later. Joint pathology was accompanied by sustained induction of IL-1ß and TNF-α in plasma over 4 weeks after administration of CII in CFA. RESULTS: CIA reduced levels of soluble and insoluble amyloid beta (Aß) peptides and amyloid plaque formation in the cortex and hippocampus of APP/PS1 mice, which correlated with increased blood brain barrier disruption, Iba-1-positive microglia, and CD45-positive microglia/macrophages. In contrast, CIA reduced vessel density and length with features of microvascular pathology, including vascular segments, thinner vessels, and atrophic string vessels. CONCLUSIONS: The present findings suggest that RA may exert beneficial effects against Aß burden and harmful effects on microvascular pathology in AD.

Iron mediates endothelial cell damage and blood-brain barrier opening in the hippocampus after transient forebrain ischemia in rats.

Blood cells are transported into the brain and are thought to participate in neurodegenerative processes following hypoxic ischemic injury. We examined the possibility that transient forebrain ischemia (TFI) causes the blood-brain barrier (BBB) to become permeable to blood cells, possibly via dysfunction and degeneration of endothelial cells in rats. Extravasation of Evans blue and immunoglobulin G (IgG) was observed in the hippocampal CA1-2 areas within 8 h after TFI, and peaked at 48 h. This extravasation was accompanied by loss of tight junction proteins, occludin, and zonula occludens-1, and degeneration of endothelial cells in the CA1-2 areas. Iron overload and mitochondrial free radical production were evident in the microvessel endothelium of the hippocampus before endothelial cell damage occurred. Administration of deferoxamine (DFO), an iron chelator, or Neu2000, an antioxidant, blocked free radical production and endothelial cell degeneration. Our findings suggest that iron overload and iron-mediated free radical production cause loss of tight junction proteins and degeneration of endothelial cells, opening of the BBB after TFI.

Chronic cerebral hypoperfusion in a mouse model of Alzheimer's disease: an additional contributing factor of cognitive impairment.

The purpose of the present study was to evaluate whether chronic cerebral hypoperfusion would affect cognitive status in an Alzheimer mouse model. Behavioral tests and histological evaluations were performed using female Tg2576 mice eight weeks after right common carotid artery occlusion (rCCAO), which is known to induce a type of vascular dementia without neuronal necrosis in nontransgenic mice. Positron emission tomography with (18)F-fluorodeoxyglucose (FDG-PET) was utilized to evaluate metabolic status in the rCCAO-operated brain of nontransgenic mice. Escape latency from the Morris water maze test was not significantly different between rCCAO- and sham-operated mice. However, the learning curve was impaired in rCCAO-operated transgenic mice while it was preserved in sham-operated transgenic or rCCAO-operated nontransgenic mice. Histological examination revealed no evidence of cell death in the rCCAO-operated brains, and the extent of amyloid deposition was not different in rCCAO- and sham-operated mice. The brain of rCCAO-operated mice showed metabolic deficits in the ipsilateral parietal cortex through FDG-PET. In conclusion, further cognitive decline which is more comparable to typical Alzheimer's disease was induced by chronic cerebral hypoperfusion in an Alzheimer mouse model. This aggravation might be associated with hypometabolism via chronic cerebral hypoperfusion.

Blood-derived iron mediates free radical production and neuronal death in the hippocampal CA1 area following transient forebrain ischemia in rat.

Abnormal brain iron homeostasis has been proposed as a pathological event leading to oxidative stress and neuronal injury under pathological conditions. We examined the possibility that neuronal iron overload would mediate free radical production and delayed neuronal death (DND) in hippocampal CA1 area after transient forebrain ischemia (TFI). Mitochondrial free radicals (MFR) were biphasically generated in CA1 neurons 0.5-8 and 48-60 h after TFI. Treatment with Neu2000, a potent spin trapping molecule, as well as trolox, a vitamin E analogue, blocked the biphasic MFR production and attenuated DND in the CA1, regardless of whether it was administered immediately or even 24 h after reperfusion. The late increase in MFR was accompanied by iron accumulation and blocked by the administration of deferoxamine-an iron chelator. Iron accumulation was attributable to prolonged upregulation of the transferrin receptor and to increased uptake of peripheral iron through a leaky blood-brain barrier. Infiltration of iron-containing cells and iron accumulation were attenuated by depletion of circulating blood cells through X-ray irradiation of the whole body except the head. The present findings suggest that excessive iron transported from blood mediates slowly evolving oxidative stress and neuronal death in CA1 after TFI, and that targeting iron-mediated oxidative stress holds extended therapeutic time window against an ischemic event.

Microglial P2X7 receptor expression is accompanied by neuronal damage in the cerebral cortex of the APPswe/PS1dE9 mouse model of Alzheimer's disease.

The possibility that P2X7 receptor (P2X7R) expression in microglia would mediate neuronal damage via reactive oxygen species (ROS) production was examined in the APPswe/PS1dE9 mouse model of Alzheimer's disease (AD). P2X7R was predominantly expressed in CD11b-immunopositive microglia from 3 months of age before Abeta plaque formation. In addition, gp91phox, a catalytic subunit of NADPH oxidase, and ethidium fluorescence were detected in P2X7R-positive microglial cells of animals at 6 months of age, indicating that P2X7R-positive microglia could produce ROS. Postsynaptic density 95-positive dendrites showed significant damage in regions positive for P2X7R in the cerebral cortex of 6 month-old mice. Taken together, up-regulation of P2X7R activation and ROS production in microglia are parallel with Aß increase and correlate with synaptotoxicity in AD.

Neu2000, an NR2B-selective, moderate NMDA receptor antagonist and potent spin trapping molecule for stroke.

Excess activation of ionotropic glutamate receptors, primarily N-methyl-D-aspartate (NMDA) receptors and free radicals, evoke nerve cell death following hypoxic-ischemic brain injury in various animal models. However, clinical trials in stroke patients using NMDA receptor antagonists have failed to show efficacy primarily due to the limited therapeutic time window for neuroprotection and a narrow therapeutic index. In comparison, antioxidants prolonged the time window for neuroprotection in animal models of ischemic stroke and showed greater therapeutic potential in clinical trials for ischemic stroke. Excess activation of NMDA receptors and free radicals mediate the two separate pathways of nerve cell death in stroke and a safe and multifunctional drug that can block both routes in the brain will likely provide a better therapeutic outcome in patients with stroke. Derivatives of the lead structures of sulfasalazine and aspirin have led to the discovery of a new molecule, Neu2000, that has demonstrated excellent neuroprotection against NMDA- and free radical-induced cell death. Neu2000 is an NR2B-selective, moderate NMDA receptor antagonist with potent cell-permeable, spin trapping antioxidant action even at nanomolar concentrations. Nonclinical and human phase I studies demonstrated that Neu2000 can be translated to treat patients with stroke with better efficacy and therapeutic time window.

Induction of the unfolded protein response and cell death pathway in Alzheimer's disease, but not in aged Tg2576 mice.

The endoplasmic reticulum (ER) stress results from disrupted protein folding triggered by protein mutation or oxidation, reduced proteasome activity, and altered Ca2+ homeostasis. ER stress is accompanied by activation of the unfolded protein response (UPR) and cell death pathway. We examined if the UPR and cell death pathway would be activated in Alzheimer's disease (AD). RT-PCR experiments revealed increased splicing of X-box binding protein-1 (XBP-1), an UPR transcription factor, in AD compared with age-matched control. Among target genes of XBP-1, expression of protein disulfide isomerase (PDI), but not glucose-regulated protein 78 (GRP78), was increased in AD, suggesting disturbed activation of the UPR in AD. C/EBP homologous protein (CHOP), caspase-3, caspase-4, and caspase-12, downstream mediators of cell death pathway, were activated in AD. Neither the UPR nor cell death pathway was induced in aged Tg2576 mice, a transgenic mouse model of Alzheimer's disease that reveals both plaque pathology and some cognitive deficits. The present study suggests that disturbed induction of the UPR and activation of the pro-apoptotic proteins contribute to neuropathological process in AD irrespective of amyloid beta and senile plaque.

Hypoxic ischemia and proteasome dysfunction alter tau isoform ratio by inhibiting exon 10 splicing.

Alternative splicing of tau exon 10 influences microtubule assembly and stability during development and in pathological processes of the central nervous system. However, the cellular events that underlie this pre-mRNA splicing remain to be delineated. In this study, we examined the possibility that ischemic injury, known to change the cellular distribution and expression of several RNA splicing factors, alters the splicing of tau exon 10. Transient occlusion of the middle cerebral artery reduced tau exon 10 inclusion in the ischemic cortical area within 12 h, resulting in the induction of three-repeat (3R) tau in cortical neurons. Ubiquitinated protein aggregates and reduced proteasome activity were also observed. Administration of proteasome inhibitors such as MG132, proteasome inhibitor I and lactacystin reduced tau exon 10 splicing in cortical cell cultures. Decreased levels of Tra2beta, an RNA splicing factor responsible for tau exon 10 inclusion, were detected both in cortical cell cultures exposed to MG132 and in cerebral cortex after ischemic injury. Taken together, these findings suggest that transient focal cerebral ischemia reduces tau exon 10 splicing through a mechanism involving proteasome-ubiquitin dysfunction and down-regulation of Tra2beta.