Mehrdad Shamloo

Abstract

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.

View details for DOI 10.1371/journal.pone.0111898

View details for PubMedID 25390368

View details for DOI 10.3233/JAD-140036

View details for Web of Science ID 000341572000012

Abstract

Stroke is the most common cause of long-term disability, and there are no known drug therapies to improve recovery after stroke. To understand how successful recovery occurs, dissect candidate molecular pathways, and test new therapies, there is a need for multiple distinct mouse stroke models, in which the parameters of recovery after stroke are well defined. Hypoxic-ischemic stroke is a well-established stroke model, but behavioral recovery in this model is not well described. We therefore examined a panel of behavioral tests to see whether they could be used to quantify functional recovery after hypoxic-ischemic stroke. We found that in C57BL/6J mice this stroke model produces high mortality (approximately one-third) and variable stroke sizes, but is fast and easy to perform on a large number of mice. Horizontal ladder test performance on day 1 after stroke was highly and reproducibly correlated with stroke size (P < 0.0001, R(2) = 0.7652), and allowed for functional stratification of mice into a group with >18% foot faults and 2.1-fold larger strokes. This group exhibited significant functional deficits for as long as 3 weeks on the horizontal ladder test and through the last day of testing on automated gait analysis (33 days), rotarod (30 days), and elevated body swing test (EBST) (36 days). No deficits were observed in an automated activity chamber. We conclude that stratification by horizontal ladder test performance on day 1 identifies a subset of mice in which functional recovery from hypoxic-ischemic stroke can be studied.

View details for DOI 10.1002/brb3.77

View details for PubMedID 23139913

Abstract

Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of 'wear and tear'. Although low-grade inflammation is detected in osteoarthritis, its role is unclear. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in complement component 5 (C5), C6 or the complement regulatory protein CD59a, we show that complement, specifically, the membrane attack complex (MAC)-mediated arm of complement, is crucial to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints from C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Further, MAC colocalized with matrix metalloprotease 13 (MMP13) and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints has a key role in the pathogenesis of osteoarthritis.

View details for DOI 10.1038/nm.2543

View details for Web of Science ID 000297978000042

View details for PubMedID 22057346

Abstract

Autism and autism spectrum disorder (ASD) typically arise from a mixture of environmental influences and multiple genetic alterations. In some rare cases, such as Timothy syndrome (TS), a specific mutation in a single gene can be sufficient to generate autism or ASD in most patients, potentially offering insights into the etiology of autism in general. Both variants of TS (the milder TS1 and the more severe TS2) arise from missense mutations in alternatively spliced exons that cause the same G406R replacement in the Ca(V)1.2 L-type calcium channel. We generated a TS2-like mouse but found that heterozygous (and homozygous) animals were not viable. However, heterozygous TS2 mice that were allowed to keep an inverted neomycin cassette (TS2-neo) survived through adulthood. We attribute the survival to lowering of expression of the G406R L-type channel via transcriptional interference, blunting deleterious effects of mutant L-type channel overactivity, and addressed potential effects of altered gene dosage by studying Ca(V)1.2 knockout heterozygotes. Here we present a thorough behavioral phenotyping of the TS2-neo mouse, capitalizing on this unique opportunity to use the TS mutation to model ASD in mice. Along with normal general health, activity, and anxiety level, TS2-neo mice showed markedly restricted, repetitive, and perseverative behavior, altered social behavior, altered ultrasonic vocalization, and enhanced tone-cued and contextual memory following fear conditioning. Our results suggest that when TS mutant channels are expressed at levels low enough to avoid fatality, they are sufficient to cause multiple, distinct behavioral abnormalities, in line with the core aspects of ASD.

View details for DOI 10.1073/pnas.1112667108

View details for Web of Science ID 000294804900085

View details for PubMedID 21878566

Abstract

Multiple independent mutations in neuroligin genes were identified in patients with familial autism, including the R451C substitution in neuroligin-3 (NL3). Previous studies showed that NL3(R451C) knock-in mice exhibited modestly impaired social behaviors, enhanced water maze learning abilities, and increased synaptic inhibition in the somatosensory cortex, and they suggested that the behavioral changes in these mice may be caused by a general shift of synaptic transmission to inhibition. Here, we confirm that NL3(R451C) mutant mice behaviorally exhibit social interaction deficits and electrophysiologically display increased synaptic inhibition in the somatosensory cortex. Unexpectedly, however, we find that the NL3(R451C) mutation produced a strikingly different phenotype in the hippocampus. Specifically, in the hippocampal CA1 region, the NL3(R451C) mutation caused an ?1.5-fold increase in AMPA receptor-mediated excitatory synaptic transmission, dramatically altered the kinetics of NMDA receptor-mediated synaptic responses, induced an approximately twofold up-regulation of NMDA receptors containing NR2B subunits, and enhanced long-term potentiation almost twofold. NL3 KO mice did not exhibit any of these changes. Quantitative light microscopy and EM revealed that the NL3(R451C) mutation increased dendritic branching and altered the structure of synapses in the stratum radiatum of the hippocampus. Thus, in NL3(R451C) mutant mice, a single point mutation in a synaptic cell adhesion molecule causes context-dependent changes in synaptic transmission; these changes are consistent with the broad impact of this mutation on murine and human behaviors, suggesting that NL3 controls excitatory and inhibitory synapse properties in a region- and circuit-specific manner.

View details for DOI 10.1073/pnas.1111093108

View details for Web of Science ID 000293895100079

View details for PubMedID 21808020

Abstract

Down syndrome (DS) is the most prevalent form of mental retardation caused by genetic abnormalities in humans. This has been successfully modeled in mice to generate the Ts65Dn mouse, a genetic model of DS. This transgenic mouse model shares a number of physical and functional abnormalities with people with DS, including changes in the structure and function of neuronal circuits. Significant abnormalities in noradrenergic (NE-ergic) afferents from the locus coeruleus to the hippocampus, as well as deficits in NE-ergic neurotransmission are detected in these animals. In the current study we characterized in detail the behavioral phenotype of Ts65Dn mice, in addition to using pharmacological tools for identification of target receptors mediating the learning and memory deficits observed in this model of DS. We undertook a comprehensive approach to mouse phenotyping using a battery of standard and novel tests encompassing: (i) locomotion (Activity Chamber, PhenoTyper, and CatWalk), (ii) learning and memory (spontaneous alternation, delayed matching-to-place water maze, fear conditioning, and Intellicage), and (iii) social behavior. Ts65Dn mice showed increased locomotor activity in novel and home cage environments. There were significant and reproducible deficits in learning and memory tests including spontaneous alternation, delayed matching-to-place water maze, Intellicage place avoidance and contextual fear conditioning. Although Ts65Dn mice showed no deficit in sociability in the 3-chamber test, a marked impairment in social memory was detected. Xamoterol, a ?1-adrenergic receptor (?1-ADR) agonist, effectively restored the memory deficit in contextual fear conditioning, spontaneous alternation and novel object recognition. These behavioral improvements were reversed by betaxolol, a selective ?1-ADR antagonist. In conclusion, our results demonstrate that this mouse model of Down syndrome displays cognitive deficits which are mediated by an imbalance in the noradrenergic system. In this experimental model of Down syndrome a selective activation of ?1-ADR does restore some of these behavioral deficits. Further mechanistic studies will be needed to investigate the failure of noradrenergic system and the role of ?1-ADR in cognitive deficit and pathogenesis of DS in people. Restoring NE neurotransmission or a selective activation of ?1)-ADR needs to be further investigated for the development of any potential therapeutic strategy for symptomatic relief of memory deficit in DS. Furthermore, due to the significant involvement of noradrenergic system in the cardiovascular function further safety and translational studies will be needed to ensure the safety and efficacy of this approach.

View details for DOI 10.1016/j.nbd.2011.04.011

View details for Web of Science ID 000292069900012

View details for PubMedID 21527343

Abstract

Male sex is an independent risk factor for long-term neurologic deficits in human preterm infants. Using a chronic, sublethal hypoxia (CSH) mouse model of preterm brain injury, we recently demonstrated acute brain volume loss with an increased male susceptibility to hippocampal volume loss and hypomyelination. We now characterize the long-term, sex-specific effects of CSH on cognition and brain growth. Neonatal mice were treated with CSH for 8 d, raised in normoxia thereafter and underwent behavioral testing at 6 wk of age. Behavioral assays sensitive to hippocampal function were chosen. CSH-treated males had impairments in associative learning, spatial memory, and long-term social memory compared with control males. In contrast, CSH-treated females were less impaired. Persistent reductions in hippocampal and cerebellar volumes were found in adult CSH-treated males, whereas regional brain volumes in adult CSH-treated females were indistinguishable from controls. Similar to human preterm infants, males exposed to hypoxia are especially vulnerable to short-term and long-term deficits in cognition and brain growth.

View details for Web of Science ID 000292015100004

View details for PubMedID 21436761

Abstract

Middle cerebral artery occlusion (MCAO) in rats is a well-studied experimental model for ischemic stroke leading to brain infarction and functional deficits. Many preclinical studies have focused on a small time window after the ischemic episode to evaluate functional outcome for screening therapeutic candidates. Short evaluation periods following injury have led to significant setbacks due to lack of information on the delayed effects of treatments, as well as short-lived and reversible neuroprotection, so called false-positive results. In this report, we evaluated long-term functional deficit for 90 days after MCAO in two rat strains with two durations of ischemic insult, in order to identify the best experimental paradigm to assess injury and subsequent recovery. Behavioral outcomes were measured pre-MCAO followed by weekly assessment post-stroke. Behavioral tests included the 18-point composite neurological score, 28-point neuroscore, rearing test, vibrissae-evoked forelimb placing test, foot fault test and the CatWalk. Brain lesions were assessed to correlate injury to behavior outcomes at the end of study. Our results indicate that infarction volume in Sprague-Dawley rats was dependent on occlusion duration. In contrast, the infarction volume in Wistar rats did not correlate with the duration of ischemic episode. Functional outcomes were not dependent on occlusion time in either strain; however, measurable deficits were detectable long-term in limb asymmetry, 18- and 28-point neuroscores, forelimb placing, paw swing speed, and gait coordination. In conclusion, these behavioral assays, in combination with an extended long-term assessment period, can be used for evaluating therapeutic candidates in preclinical models of ischemic stroke.

View details for DOI 10.1016/j.jneumeth.2011.01.010

View details for Web of Science ID 000290781200003

View details for PubMedID 21256866

Abstract

Stroke leads to brain damage with subsequent slow and incomplete recovery of lost brain functions. Enriched housing of stroke-injured rats provides multi-modal sensorimotor stimulation, which improves recovery, although the specific mechanisms involved have not been identified. In rats housed in an enriched environment for two weeks after permanent middle cerebral artery occlusion, we found increased sigma-1 receptor expression in peri-infarct areas. Treatment of rats subjected to permanent or transient middle cerebral artery occlusion with 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride, an agonist of the sigma-1 receptor, starting two days after injury, enhanced the recovery of lost sensorimotor function without decreasing infarct size. The sigma-1 receptor was found in the galactocerebroside enriched membrane microdomains of reactive astrocytes and in neurons. Sigma-1 receptor activation increased the levels of the synaptic protein neurabin and neurexin in membrane rafts in the peri-infarct area, while sigma-1 receptor silencing prevented sigma-1 receptor-mediated neurite outgrowth in primary cortical neuronal cultures. In astrocytic cultures, oxygen and glucose deprivation induced sigma-1 receptor expression and actin dependent membrane raft formation, the latter blocked by sigma-1 receptor small interfering RNA silencing and pharmacological inhibition. We conclude that sigma-1 receptor activation stimulates recovery after stroke by enhancing cellular transport of biomolecules required for brain repair, thereby stimulating brain plasticity. Pharmacological targeting of the sigma-1 receptor provides new opportunities for stroke treatment beyond the therapeutic window of neuroprotection.

View details for DOI 10.1093/brain/awq367

View details for Web of Science ID 000287745100010

View details for PubMedID 21278085

Abstract

Cell transplantation offers a novel therapeutic strategy for stroke; however, how transplanted cells function in vivo is poorly understood. We show for the first time that after subacute transplantation into the ischemic brain of human central nervous system stem cells grown as neurospheres (hCNS-SCns), the stem cell-secreted factor, human vascular endothelial growth factor (hVEGF), is necessary for cell-induced functional recovery. We correlate this functional recovery to hVEGF-induced effects on the host brain including multiple facets of vascular repair and its unexpected suppression of the inflammatory response. We found that transplanted hCNS-SCns affected multiple parameters in the brain with different kinetics: early improvement in blood-brain barrier integrity and suppression of inflammation was followed by a delayed spatiotemporal regulated increase in neovascularization. These events coincided with a bimodal pattern of functional recovery, with, an early recovery independent of neovascularization, and a delayed hVEGF-dependent recovery coincident with neovascularization. Therefore, cell transplantation therapy offers an exciting multimodal strategy for brain repair in stroke and potentially other disorders with a vascular or inflammatory component.

View details for DOI 10.1002/stem.584

View details for Web of Science ID 000287698600011

View details for PubMedID 21732485

Abstract

Cell transplantation offers a novel therapeutic strategy for stroke; however, how transplanted cells function in vivo is poorly understood. We show for the first time that after sub-acute transplantation into the ischemic brain of human central nervous system stem cells grown as neurospheres (hCNS-SCns), the stem cell-secreted factor, human VEGF (hVEGF), is necessary for cell-induced functional recovery. We correlate this functional recovery to hVEGF-induced effects on the host brain including multiple facets of vascular repair, and its unexpected suppression of the inflammatory response. We found that transplanted hCNS-SCns affected multiple parameters in the brain with different kinetics: early improvement in blood-brain barrier (BBB) integrity and suppression of inflammation was followed by a delayed spatio-temporal regulated increase in neovascularization. These events coincided with a bi-modal pattern of functional recovery: an early recovery independent of neovascularization, and a delayed hVEGF-dependent recovery coincident with neovascularization. Therefore, cell transplantation therapy offers an exciting multi-modal strategy for brain repair in stroke and potentially other disorders with a vascular or inflammatory component.

View details for PubMedID 21240943

Abstract

Various proteases in the brain contribute to ischemic brain injury. We investigated the involvement of the asparaginyl endopeptidase legumain after experimental stroke. On the basis of gene array studies and in situ hybridizations, we observed an increase of legumain expression in the peri-infarct area of rats after transient occlusion of the middle cerebral artery (MCAO) for 120?mins with a maximum expression at 24 and 48?h. Immunohistochemical analyses revealed the expression of legumain in Iba1(+) microglial cells and glial fibrillary acidic protein-positive astrocytes of the peri-infarct area in mice after MCAO. Post-stroke recovery was also studied in aged legumain-deficient mice (45 to 58 weeks old). Legumain-deficient mice did not show any differences in physiologic parameters compared with respective littermates before, during MCAO (45?mins), and the subsequent recovery period of 8 days. Moreover, legumain deficiency had no effect on mortality, infarct volume, and the neurologic deficit determined by the rotating pole test, a standardized grip strength test, and the pole test. However, a reduced number of invading CD74(+) cells in the ischemic hemisphere indicates an involvement in post-stroke inflammation. We conclude that legumain is not essential for the functional deficit after MCAO but may be involved in mechanisms of immune cell invasion.

View details for DOI 10.1038/jcbfm.2010.39

View details for Web of Science ID 000282382200009

View details for PubMedID 20234379

Abstract

Major histocompatibility complex class I (MHCI) genes were discovered unexpectedly in healthy CNS neurons in a screen for genes regulated by neural activity. In mice lacking just 2 of the 50+ MHCI genes H2-K(b) and H2-D(b), ocular dominance (OD) plasticity is enhanced. Mice lacking PirB, an MHCI receptor, have a similar phenotype. H2-K(b) and H2-D(b) are expressed not only in visual cortex, but also in lateral geniculate nucleus (LGN), where protein localization correlates strongly with synaptic markers and complement protein C1q. In K(b)D(b-/-) mice, developmental refinement of retinogeniculate projections is impaired, similar to C1q(-/-) mice. These phenotypes in K(b)D(b-/-) mice are strikingly similar to those in beta2 m(-/-)TAP1(-/-) mice, which lack cell surface expression of all MHCIs, implying that H2-K(b) and H2-D(b) can account for observed changes in synapse plasticity. H2-K(b) and H2-D(b) ligands, signaling via neuronal MHCI receptors, may enable activity-dependent remodeling of brain circuits during developmental critical periods.

View details for DOI 10.1016/j.neuron.2009.10.015

View details for Web of Science ID 000272378900005

View details for PubMedID 19945389

Abstract

Down syndrome (trisomy 21) is the most common cause of mental retardation in children and leads to marked deficits in contextual learning and memory. In rodents, these tasks require the hippocampus and are mediated by several inputs, particularly those originating in the locus coeruleus. These afferents mainly use norepinephrine as a transmitter. To explore the basis for contextual learning defects in Down syndrome, we examined the Ts65Dn mouse model. These mice, which have three copies of a fragment of mouse chromosome 16, exhibited significant deficits in contextual learning together with dysfunction and degeneration of locus coeruleus neurons. However, the postsynaptic targets of innervation remained responsive to noradrenergic receptor agonists. Indeed, despite advanced locus coeruleus degeneration, we were able to reverse contextual learning failure by using a prodrug for norepinephrine called l-threo-3,4-dihydroxyphenylserine, or xamoterol, a beta(1)-adrenergic receptor partial agonist. Moreover, an increased gene dosage of App, in the context of Down syndrome, was necessary for locus coeruleus degeneration. Our findings raise the possibility that restoring norepinephrine-mediated neurotransmission could reverse cognitive dysfunction in Down syndrome.

View details for DOI 10.1126/scitranslmed.3000258

View details for Web of Science ID 000277262200002

View details for PubMedID 20368182

Abstract

Striatal enriched protein tyrosine phosphatase (STEP) acts in the central nervous system to dephosphorylate a number of important proteins involved in synaptic function including ERK and NMDA receptor subunits. These proteins are also linked to stroke, in which cerebral ischemia triggers a complex cascade of events. Here we demonstrate that STEP is regulated at both the transcriptional and the post-transcriptional levels in rat models of cerebral ischemia and that its regulation may play a role in the outcome of ischemic insults. After transient middle cerebral artery occlusion, there are profound decreases in the levels of STEP mRNA, whilst in global ischemia STEP mRNA is selectively down-regulated in areas susceptible to ischemic damage. In a neuroprotective preconditioning paradigm, and in regions of the brain that are relatively resistant to ischemic damage, STEP mRNA levels are increased. Furthermore, there is a significant processing of STEP after ischemia to generate a novel species, STEP(33), resulting in a redistribution of STEP from membrane-bound to soluble compartments. Concomitant with the cleavage of mature forms of STEP, there are changes in the phosphorylation state of ERK. We show that the cleavage of STEP leads to a catalytically active form, but this cleaved form no longer binds to and dephosphorylates its substrate pERK. Therefore, in response to ischemic insults, there are profound reductions in both the amount and the activity of STEP, its localization, as well as the activity of one of its key substrates, pERK. These changes in STEP may reflect a critical role in the outcomes of ischemic brain injury.

View details for DOI 10.1111/j.1460-9568.2008.06209.x

View details for Web of Science ID 000255285800024

View details for PubMedID 18445231

Abstract

Basic helix-loop-helix PAS domain proteins form a growing family of transcription factors. These proteins are involved in the process of adaptation to cellular stresses and environmental factors such as a change in oxygen concentration. We describe the identification and characterization of a recently cloned PAS domain protein termed Npas4 in ischemic rat brain. Using gene expression profiling following middle cerebral artery occlusion, we showed that the Npas4 mRNA is differentially expressed in ischemic tissue. The full-length gene was cloned from rat brain and its spatial and temporal expression characterized with in situ hybridization and Northern blotting. The Npas4 mRNA is specifically expressed in the brain and is highly up-regulated in ischemic tissues following both focal and global cerebral ischemic insults. Immunohistochemistry revealed a strong expression in the limbic system and thalamus, as well as in layers 3 and 5 in the cortex of the unchallenged brain. When overexpressed in HEK 293 cells, Npas4 appears as a protein of approximately 100 kDa. In brain samples, however, in addition to the 100 kDa band a specific 200 kDa immunoreactive band was also detected. Ischemic challenge lead to a decrease in the 200 kDa form and a simultaneous increase in the 100 kDa immunoreactivity. This could indicate a novel regulatory mechanism for activation and/or deactivation of this protein in response to ischemic brain injury.

View details for DOI 10.1111/j.1460-9568.2006.05172.x

View details for Web of Science ID 000242221700003

View details for PubMedID 17156197

Abstract

In order to identify biological processes relevant for cell death and survival in the brain following stroke, the postischemic brain transcriptome was studied by a large-scale cDNA array analysis of three peri-infarct brain regions at eight time points during the first 24 h of reperfusion following middle cerebral artery occlusion in the rat. K-means cluster analysis revealed two distinct biphasic gene expression patterns that contained 44 genes (including 18 immediate early genes), involved in cell signaling and plasticity (i.e. MAP2K7, Sprouty2, Irs-2, Homer1, GPRC5B, Grasp). The first gene induction phase occurred at 0-3 h of reperfusion, and the second at 9-15 h, and was validated by in situ hybridization. Four gene clusters displayed a progressive increase in expression over time and included 50 genes linked to cell motility, lipid synthesis and trafficking (i.e. ApoD, NPC1, G3P-dehydrogenase1, and Choline kinase) or cell death-regulating genes such as mitochondrial CLIC. We conclude that a biphasic transcriptional up-regulation of the brain-derived neurotrophic factor (BDNF)-G-protein coupled receptor (GPCR)-mitogen-activated protein (MAP) kinase signaling pathways occurs in surviving tissue, concomitant with a progressive and persistent activation of cell proliferation signifying tissue regeneration, which provide the means for cell survival and postischemic brain plasticity.

View details for DOI 10.1111/j.1471-4159.2005.03508.x

View details for Web of Science ID 000233829900002

View details for PubMedID 16300643

Abstract

Death-associated protein kinase (DAPK) is a calcium calmodulin-regulated serine/threonine protein kinase involved in ischemic neuronal death. In situ hybridization experiments show that DAPK mRNA expression is up-regulated in brain following a global ischemic insult and down-regulated in ischemic tissues after focal ischemia. DAPK is inactive in normal brain tissues, where it is found in its phosphorylated state and becomes rapidly and persistently dephosphorylated and activated in response to ischemia in vivo. A similar dephosphorylation pattern is detected in primary cortical neurons subjected to oxygen glucose deprivation or N-methyl-D-aspartate (NMDA)-induced toxicity. Both a calcineurin inhibitor, FK506, and a selective NMDA receptor antagonist, MK-801, inhibit the dephosphorylation of DAPK after in vitro ischemia. This indicates that DAPK could be activated by NMDA receptor-mediated calcium flux, activation of calcineurin, and subsequent DAPK dephosphorylation. Moreover, concomitantly to dephosphorylation, DAPK is proteolytically processed by cathepsin after ischemia. Furthermore, a selective DAPK inhibitor is neuroprotective in both in vitro and in vivo ischemic models. These results indicate that DAPK plays a key role in mediating ischemic neuronal injury.

View details for DOI 10.1074/jbc.M505804200

View details for Web of Science ID 000233992700058

View details for PubMedID 16204252

Abstract

The levels of protein kinase C-gamma (PKC-gamma ) and the calcium/calmodulin-dependent kinase II-alpha (CaMKII-alpha) were measured in crude synaptosomal (P2), particulate (P3), and cytosolic (S3) fractions of the neocortex of rats exposed to 1-hour and 2-hour middle cerebral artery occlusion (MCAO) and 2-hour MCAO followed by 2-hour reperfusion. During MCAO, PKC levels increased in P2 and P3 in the most severe ischemic areas concomitantly with a decrease in S3. In the penumbra, PKCgamma decreased in S3 without any significant increases in P2 and P3. Total PKC-gamma also decreased in the penumbra but not in the ischemic core, suggesting that the protein is degraded by an energy-dependent mechanism, possibly by the 26S proteasome. The CaMKII-alpha levels increased in P2 but not P3 during ischemia and reperfusion in all ischemic regions, particularly in the ischemic core. Concomitantly, the levels in S3 decreased by 20% to 40% in the penumbra and by approximately 80% in the ischemic core. There were no changes in the total levels of CaMKII-alpha during MCAO. The authors conclude that during and after ischemia, PKC and CaMKII-alpha are translocated to the cell membranes, particularly synaptic membranes, where they may modulate cellular function, such as neurotransmission, and also affect cell survival. Drugs preventing PKC and/or CaMKII-alpha translocation may prove beneficial against ischemic cell death.

View details for DOI 10.1097/01.WCB.0000095920.70924.F5

View details for Web of Science ID 000187577500006

View details for PubMedID 14688616

Abstract

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.

View details for DOI 10.1038/nm903

View details for Web of Science ID 000184484900031

View details for PubMedID 12858170

Abstract

Transient cerebral ischemia following 1 to 2 hours of middle cerebral artery occlusion (MCAO) in the rat leads to infarction, which can be diminished by synaptic transmission modulators, implying aberrant cell signaling in the pathogenetic process. The authors report here changes in the levels of tyrosine phosphorylated proteins (PTyr) and calcium calmodulin kinase II (CaMKII) phosphorylation of Thr 286, in synaptosomal, particulate, and cytosolic fractions of different cortical areas following 1 or 2 hours of MCAO, or 2 hours of MCAO followed by 2 hours of reperfusion. At the end of 2-hour MCAO, PTyr, and in particular the pp180, indicative of NR2A/B subunit, increased in the synaptosomal fraction in less ischemic areas while it decreased in more severe ischemic regions. During reperfusion, phosphorylation increased at least 2-fold in all reperfused areas. During 2 hours of MCAO, the phosphorylation of CaMKII increased 8- to 10-fold in the synaptosomal fraction in all ischemic brain regions. During reperfusion, the phospho-CaMKII levels remained elevated by approximately 300% compared with the contralateral hemisphere (control). There was no increase in phospho-CaMKII in the cytosolic fraction at any time during or following ischemia in any of the brain regions examined. The authors conclude that both tyrosine kinase coupled pathways, as well as CaMKII-mediated cellular processes associated with synaptic activity, are strongly activated during and particularly following MCAO. These results support the hypothesis that aberrant cell signaling may contribute to ischemic cell death and dysfunction, and that selective modulators of cell signaling may be targets for pharmacological intervention against ischemic brain damage.

View details for Web of Science ID 000177919400008

View details for PubMedID 12218416

Abstract

A brief period of sublethal ischemia induces resistance to a subsequent, otherwise lethal, ischemic insult, a process named ischemic tolerance or preconditioning. A persistently disturbed cell signaling during reperfusion after cerebral ischemia has been proposed to contribute to ischemic cell death. Here, we report on the effect of ischemic preconditioning on the levels of the regulatory alpha-subunit of calcium/calmodulin protein kinase II and its phosphorylation in the hippocampal CA1 region. We found that during and following lethal cerebral ischemia, calcium/calmodulin protein kinase II-alpha is persistently translocated to cell membranes, where it becomes phosphorylated at threonine 286. In contrast, in the preconditioned brains the translocation and phosphorylation are transient and return to preischemic values after one day of reperfusion. At this time of reperfusion, the total level of calcium/calmodulin protein kinase II-alpha is significantly lower in preconditioned animals compared to the sham and non-conditioned animals. After one day of reperfusion, the level of calcium/calmodulin protein kinase II-alpha messenger RNA decreases in the non-conditioned brains, whereas it is unchanged in preconditioned brains. We conclude that, during and after ischemia, calcium/calmodulin protein kinase II-alpha is translocated to cell membranes and becomes phosphorylated at threonine 286. This could detrimentally influence cell survival by changing receptor function and ion channel conductance. Ischemic preconditioning prevents the persistent presence of calcium/calmodulin protein kinase II-alpha at cell membranes, presumably by enhancing its degradation, which could be part of a neuroprotective mechanism of ischemic tolerance.

View details for Web of Science ID 000086204000003

View details for PubMedID 10727785

Abstract

A brief, 3 min period of global forebrain ischemia in the rat, induced by bilateral common carotid occlusion combined with hypotension, confers resistance to hippocampal pyramidal neurons against a subsequent 10 min ischemia, which is normally lethal to these cells. The molecular mechanisms underlying this ischemic preconditioning, or tolerance, are poorly understood. The tumor suppressor p53 is a transcription factor implicated in neuronal death following various insults, including cerebral ischemia. p53 is activated in response to cellular stress, e.g. hypoxia and DNA damage. Using in situ hybridization, we investigated the hippocampal mRNA expression of p53, and two of its target genes, p21(WAF1/Cip1) and the recently cloned PAG608/Wig-1, in a two-vessel occlusion model of ischemic preconditioning. We also evaluated changes in the protein levels of p53 and PAG608/Wig-1 using immunohistochemistry. The mRNA levels of all three genes increased in the ischemia sensitive CA1 region both following 3 min (non-lethal) preconditioning and 10 min of (lethal) nonconditioned ischemia. In contrast, after 10 min of ischemia preconditioned by a 3 min ischemic insult 48 h earlier, no upregulation of these genes was detected in the CA1. Following 10 min of nonconditioned ischemia, increased neuronal immunostaining of p53 and PAG608/Wig-1 was observed in the hippocampus, which was less pronounced following 3 min of preconditioning ischemia and 10 min of preconditioned ischemia. Our results demonstrate that activation of p53 and its response genes p21(WAF1/Cip1) and PAG608/Wig-1 occurs in the brain following lethal as well as non-lethal ischemic insults, and that ischemic preconditioning markedly diminishes this activation.

View details for Web of Science ID 000081514900015

View details for PubMedID 10407180

Abstract

Neurons can be preconditioned against ischemic damage by a brief sublethal period of ischemia, applied several days before the second insult. Here we report on changes in the distribution and the levels of protein kinase Cgamma (PKCgamma) in nonconditioned and preconditioned rat hippocampal CA1 and neocortex regions after a 9 min ischemic episode induced by two-vessel occlusion ischemia. At the end of the second ischemia we found significantly lower levels of PKCgamma in the CA1 region but not neocortex of preconditioned brains than in non-conditioned brains. Protein kinase Cgamma levels in both CA1 and neocortex decrease simultaneously in the cytosolic fractions. We conclude that PKCgamma is translocated to cell membranes during ischemia and is rapidly removed or degraded during the second otherwise lethal ischemic insult in preconditioned brains. The data suggest that ischemic preconditioning enhances downregulation of cell signaling mediated by PKCgamma and may thereby provide neuroprotection.

View details for Web of Science ID 000079904900009

View details for PubMedID 10321462

Abstract

A brief period of sublethal cerebral ischemia, followed by several days of recovery, renders the brain resistant to a subsequent lethal ischemic insult, a phenomenon termed ischemic preconditioning or tolerance. Ischemic tolerance was established in the rat two-vessel occlusion model of ischemia, induced by occlusion of both carotid arteries in combination with hypotension. Ischemic preconditioning (3 minutes) provided maximal neuroprotection when induced 2 days prior to a lethal ischemic insult of 9-minute duration. Neuroprotection persisted for at least 8 weeks. Since neurotransmission has been implicated in ischemic cell death, the effect of ischemic preconditioning on tyrosine phosphorylation of proteins and on the levels of glutamate receptor subunits in hippocampus and neocortex was studied. Regional levels of tyrosine phosphorylation of proteins in general and the N-methyl-D-aspartate receptor subunit NR2 in particular are markedly enhanced after ischemia in nonconditioned brains, in both the synaptosomal fraction and the whole-tissue homogenate of rat neocortex and hippocampus, but recover to control levels only in the preconditioned brain. Ischemic preconditioning selectively induces a decrease in the levels of the NR2A and NR2B subunits and a modest decrease in the levels of NR1 subunit proteins in the synaptosomal fraction of the neocortex but not hippocampus after the second lethal ischemia. It was concluded that ischemic preconditioning prevents a persistent change in cell signaling as evidenced by the tyrosine phosphorylation of proteins after the second lethal ischemic insult, which may abrogate the activation of detrimental cellular processes leading to cell death.

View details for Web of Science ID 000079669300009

View details for PubMedID 10027773

Abstract

A short period of sublethal preconditioning ischemia (3 min) followed by two days of reperfusion provides almost complete protection against ischemic cell death induced by a second (9 min) lethal ischemic episode. Here, we have investigated the extracellular signal-regulated protein kinase kinase and extracellular signal-regulated protein kinase, two kinases known to activate gene transcription and to be of importance for cell survival, after sublethal preconditioning ischemia in the rat hippocampal CA1 region. The activation levels of these two kinases were also studied after a second ischemic episode both in preconditioned and nonconditioned brains. An increased phosphorylation of the extracellular signal-regulated protein kinase kinase was found in neuronal cell bodies, particularly in the nucleus, 30 min, 4 h and two days of reperfusion after preconditioning ischemia. Two days after preconditioning ischemia both extracellular signal-regulated protein kinase kinase and extracellular signal-regulated protein kinase were markedly phosphorylated. During the early reperfusion period (30 min) after the second ischemic insult the phosphorylation levels of these two kinases were increased in both nonconditioned and preconditioned brains. In the late reperfusion time (one day), the phosphorylation levels of the extracellular signal-regulated protein kinase kinase and extracellular signal-regulated protein kinase were decreased in preconditioned brains, but remained elevated in nonconditioned brains. We conclude that phosphorylation of the extracellular signal-regulated protein kinase kinase and extracellular signal-regulated protein kinase after sublethal ischemia correlates with the neuroprotection induced by preconditioning, possibly by transcriptional activation of neuroprotective genes. Also, preconditioning enhances normalization of the disturbed cell signaling through the extracellular signal-regulated protein kinase cascade induced by lethal ischemia.

View details for Web of Science ID 000081196200007

View details for PubMedID 10430472

Abstract

The regional selectivity and mechanisms underlying the toxicity of the serine/threonine protein phosphatase inhibitor okadaic acid (OA) were investigated in hippocampal slice cultures. Image analysis of propidium iodide-labeled cultures revealed that okadaic acid caused a dose- and time-dependent injury to hippocampal neurons. Pyramidal cells in the CA3 region and granule cells in the dentate gyrus were much more sensitive to okadaic acid than the pyramidal cells in the CA1 region. Electron microscopy revealed ultrastructural changes in the pyramidal cells that were not consistent with an apoptotic process. Treatment with okadaic acid led to a rapid and sustained tyrosine phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2 (p44/42(mapk)). The phosphorylation was markedly reduced after treatment of the cultures with the microbial alkaloid K-252a (a nonselective protein kinase inhibitor) or the MAP kinase kinase (MEK1/2) inhibitor PD98059. K-252a and PD98059 also ameliorated the okadaic acid-induced cell death. Inhibitors of protein kinase C, Ca2+/calmodulin-dependent protein kinase II, or tyrosine kinase were ineffective. These results indicate that sustained activation of the MAP kinase pathway, as seen after e.g., ischemia, may selectively harm specific subsets of neurons. The susceptibility to MAP kinase activation of the CA3 pyramidal cells and dentate granule cells may provide insight into the observed relationship between cerebral ischemia and dementia in Alzheimer's disease.

View details for Web of Science ID 000075893000023

View details for PubMedID 9736650

Abstract

Rat and gerbil hippocampus exposed to a sublethal period of ischemia becomes resistant to a subsequent period of lethal ischemia induced several days later, a phenomenon referred to as ischemic preconditioning. Here we describe ischemic preconditioning induced in vitro in cultured hippocampal neurons. Mixed neuroglial hippocampal cell cultures of 14-17 DIV were exposed to a combined glucose and oxygen deprivation (GOD). Cultures subjected to 90 min, but not 60 min, of GOD showed extensive degeneration after a 1 day recovery period. An episode of 60 min of preconditioning GOD followed 1 and 2 days later by 90 min of GOD resulted in 40-60% protection. The data demonstrate that ischemic preconditioning can be mimicked in an in vitro hippocampal cell culture system.

View details for Web of Science ID 000074000000004

View details for PubMedID 9631411

Abstract

The objective of this study was to explore whether preischemic hyperglycemia, which is known to aggravate brain damage due to transient global or forebrain ischemia of intermediate duration (10 to 20 minutes), increases the density of selective neuronal necrosis, as observed primarily in the CA1 sector of the hippocampus after brief periods of forebrain ischemia in rats (2.5 and 5 minutes).Anesthetized rats were subjected to two-vessel forebrain ischemia of 2.5- or 5-minute duration. Normoglycemic or hyperglycemic rats were either allowed a recovery period of 7 days for histopathological evaluation of neuronal necrosis in the hippocampus, isocortex, thalamus, and substantia nigra or were used for recording of extracellular concentrations of Ca2+ ([Ca2+]c), K+, or H+, together with the direct current (DC) potential.Ischemia of 2.5- or 5-minute duration gave rise to similar damage in the CA1 sector of the hippocampus in normoglycemic and hyperglycemic groups (10% to 15% and 20% to 30% of the total population, respectively). However, in hyperglycemic animals subjected to 2.5 minutes of ischemia, CA1 neurons never depolarized and [Ca2+]c did not decrease. In the 5-minute groups, the total period of depolarization was 2 to 3 minutes shorter in hyperglycemic than in normoglycemic groups. This fact and results showing neocortical, thalamic, and substantia nigra damage in hyperglycemic animals after 5 minutes of ischemia demonstrate that although hyperglycemia delays the onset of ischemic depolarization and hastens repolarization and extrusion of Ca2+, it aggravates neuronal damage due to ischemia.These results reinforce the concept that hyperglycemia exaggerates brain damage due to transient ischemia and prove that this exaggeration is observed at the neuronal level. The results also suggest that the concept of the duration of an ischemic transient should be qualified, particularly if ischemia is brief, ie. < 10 minutes in duration.

View details for Web of Science ID A1996VF03200027

View details for PubMedID 8784135

Abstract

The primary objective of this study was to explore why preischemic hypoglycemia, which restricts tissue acidosis during the ischemic insult, does not ameliorate cell damage incurred as a result of transient ischemia. The question arose whether hypoglycemia (plasma glucose concentration 2-3 mM) delays resumption of extrusion of Ca2+ from cells during recirculation. Measurements of extracellular Ca2+ concentration during forebrain ischemia of 15 min duration proved that this was the case. Thus, normoglycemic animals resumed Ca2+ extrusion upon recirculation after a delay of 1.5-2.0 min, and hypoglycemic ones after an additional delay which could amount to 3-4 min. We attempted to explore the cause of this delay. At first sight, the results suggested that resumption of oxidative phosphorylation upon recirculation was substrate limited. However, glucose infusion during ischemia or just after recirculation failed to accelerate Ca2+ extrusion from the cells. A comparison between non-injected and insulin-injected animals at equal plasma glucose concentrations suggested that insulin was responsible for the delay. On analysis, the delay proved to be related to a sluggish recovery of cerebral blood flow. The results suggest that when cell damage is evaluated after transient ischemia in hypo- and normoglycemic subjects, attention should be directed to the period of cell calcium 'overload'. Unobserved differences in the duration of the calcium transient may also confound interpretation of data on the effects of insulin.

View details for Web of Science ID A1995RT05500003

View details for PubMedID 7498390

Abstract

The objective of the present experiments was to characterize conditions under which pre-ischaemic hyperglycaemia aggravates brain damage following transient forebrain ischaemia. Specifically, we wished to explore whether accentuated damage is a threshold function of plasma glucose concentration or pH, as assessed by measurements of extracellular pH (pHe). Forebrain ischaemia of 10 min duration was induced in rats at varying degrees of hyperglycaemia, with continuous measurements of pHe, and the animals were allowed to survive for 7 days before histopathological evaluation of the density and distribution of brain damage. Ischaemic brain damage appeared as a threshold function of plasma glucose concentration. At values of 4-6 mM virtually no damage was observed in any other structure than the CA1 sector of the hippocampus and, even in that structure, damage was variable. At glucose concentrations of 8-12 mM moderate damage was observed infrequently in caudoputamen, parietal cortex, and thalamus. At values above 12 mM, damage increased dramatically in these areas, and additional structures were recruited in the damage process (cingulate cortex, the CA3 sector of the hippocampus, and substantia nigra). Measurements of pHe in parietal cortex showed a threshold for seizure induction at values of 6.4-6.5, probably corresponding to intracellular pH values of 6.2-6.3. The threshold for aggravation of histopathological damage was similar. It is concluded that a moderate increase in plasma glucose in the threshold range predisposes the tissue to aggravated damage, probably by activating biochemical reactions or pathophysiological events with a steep pH dependence.

View details for Web of Science ID A1995TA63500004

View details for PubMedID 8980013

Abstract

To investigate whether aggravation of damage in hyperglycemic subjects is a continuous function of changes in intra- and extracellular pH during ischemia or whether there is a threshold value, preischemic plasma glucose was varied from 8.3-20.0 mM. 10 min forebrain ischemia was induced. The results showed that no animal with plasma glucose of < 13 mM developed seizures, and that all animals with glucose of > 16 mM died in status epilepticus. Half of the animals with plasma glucose in the range of 13-16 mM showed seizures and 50% of these died. In surviving animals, histological brain damage occurred in the hippocampal CA3 sector, cingulate cortex, thalamic nuclei and substantia nigra, structures normally not injured by 10 min ischemia. The data demonstrate that there is a glucose threshold of 10-13 mM, above which seizures develop and additional damage appears, and another one (> 16 mM), above which seizures are invariably fatal.

View details for Web of Science ID A1994PE81000016

View details for PubMedID 7824184