Charlie S. Thompson

Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus.

We show here that transient forebrain ischemia selectively elevates levels of neuronal apoptosis inhibitory protein (NAIP) in rat neurons that are resistant to the injurious effects of this treatment. This observation suggests that increasing NAIP levels may confer protection against ischemic cell death. Consistent with this proposal, we demonstrate that two other treatments that increase neuronal NAIP levels, systemic administration of the bacterial alkaloid K2S2a and intracerebral injection of an adenovirus vector capable of overexpressing NAIP in vivo, reduce ischemic damage in the rat hippocampus. Taken together, these findings suggest that NAIP may play a key role in conferring resistance to ischemic damage and that treatments that elevate neuronal levels of this antiapoptotic protein may have utility in the treatment of stroke.

APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death

p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5–6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53–DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter–luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.

ATP release by way of connexin 36 hemichannels mediates ischemic tolerance in vitro

Spreading depression (SD) is a self-propagating wave of neuronal and glial depolarization that may occur in virtually any gray matter region in the brain. One consequence of SD is an increased tolerance to ischemia. It has been shown that during cortical SD ATP is released into the extracellular space and activation of purinergic receptors leads to the induction of ischemic tolerance. In the present study we show that depolarization of cultured neurons induces ischemic tolerance which is mediated by purinergic receptor activation. Depolarization causes the release of ATP into the extracellular medium, which may be prevented by treatment with the connexin hemichannel blockers flufenamic acid and quinine, but not the pannexin hemichannel blocker carbenoxolone. Knockdown of connexin 36 expression by siRNA greatly reduces the amount of ATP released during depolarization and the subsequent degree of ischemic tolerance. We conclude that during depolarization neurons release ATP by way of connexin 36 hemichannels.

Cortical spreading depression releases ATP into the extracellular space and purinergic receptor activation contributes to the induction of ischemic tolerance

Cortical Spreading Depression (CSD) is a well-studied model of preconditioning that provides a high degree of tolerance to a subsequent ischemic event in the brain. The present study was undertaken in order to determine whether the release of ATP during CSD could contribute to the induction of ischemic tolerance. Direct measurement of ATP levels during CSD indicates that with each CSD wave ATP is released into the extracellular space at levels exceeding 100 microM. Cultures of rat primary cortical neurons exposed to low levels of extracellular ATP developed tolerance to subsequent oxygen-glucose deprivation (OGD) or metabolic hypoxia. The preconditioning effect requires new protein synthesis and develops with time, suggesting that a complex genomic response is required for the induction of tolerance. Multiple purinergic receptors are involved in mediating tolerance, with P2Y receptor activation having the greatest effect. Although extracellular adenosine or glutamate may make a small contribution, most of the tolerance was found to be induced independently of adenosine or glutamate receptor activation. Multiple signal transduction pathways mediate the response to extracellular ATP with the protein kinase A pathway and activation of phospholipase C contributing the most. The results are consistent with the proposal that CSD releases ATP into the extracellular space and the subsequent activation of P2Y receptors makes a major contribution to the induction of ischemic tolerance in the brain.

NAIP protects the nigrostriatal dopamine pathway in an intrastriatal 6-OHDA rat model of Parkinson's disease

Parkinsons disease (PD) is a progressive neurodegenerative disorder of the basal ganglia, associated with the inappropriate death of dopaminergic neurons of the substantia nigra pars compacta (SNc). Here, we show that adenovirally mediated expression of neuronal apoptosis inhibitor protein (NAIP) ameliorates the loss of nigrostriatal function following intrastriatal 6‐OHDA administration by attenuating the death of dopamine neurons and dopaminergic fibres in the striatum. In addition, we also addressed the role of the cysteine protease caspase‐3 activity in this adult 6‐OHDA model, because a role for caspases has been implicated in the loss of dopamine neurons in PD, and because NAIP is also a reputed inhibitor of caspase‐3. Although caspase‐3‐like proteolysis was induced in the SNc dopamine neurons of juvenile rats lesioned with 6‐OHDA and in adult rats following axotomy of the medial forebrain bundle, caspase‐3 is not induced in the dopamine neurons of adult 6‐OHDA‐lesioned animals. Taken together, these results suggest that therapeutic strategies based on NAIP may have potential value for the treatment of PD.

Attenuation of MPTP-induced neurotoxicity and behavioural impairment in NSE-XIAP transgenic mice.

X-linked IAP protein is a potent inhibitor of cell death. Here, we describe a novel transgenic mouse in which the human XIAP gene is expressed under the control of the neuron-specific enolase promoter (NSE-xiap). We demonstrate that nigrostriatal dopamine neurons of NSE-xiap mice were resistant to the damaging effects of the dopaminergic neurotoxin MPTP. MPTP-induced reduction of striatal dopamine metabolism was also attenuated in NSE-xiap mice. Furthermore, NSE-xiap mice treated with MPTP did not exhibit deficits in exploratory behaviour in an open-field test. Taken together, these findings suggest that strategies to enhance neuronal expression of XIAP may provide therapeutic benefit for the treatment of neurodegeneration in Parkinsons disease.

Regulation of nestin expression after cortical ablation in adult rat brain

During embryogenesis, transient expression of nestin in proliferating neuroepithelial stem cells signals the commitment of progenitor cells to differentiate. Although adult mammalian brain contains very little nestin, significant upregulation of nestin has been reported following cerebral injury, leading to speculation that nestin may be involved in brain repair. In this study, we assessed the temporal profile of nestin expression following ablation injury of the sensory barrel cortex and investigated the influence of contralateral whisker stimulation on nestin expression. Since the adult mammalian brain contains proliferating neuronal progenitor cells that can be labeled with bromodeoxyuridine (BrdU), we also determined the association of nestin reexpression with BrdU-labeled cells. Nestin reexpression was detected predominantly in the ipsilateral cortex 3 days post-ablation. There was no significant nestin upregulation in the subcortical region. Nestin reexpression was most marked surrounding the lesion, but also extended throughout the entire lateral cortex. Nestin in the ipsilateral cortex subsided by day 7, although perilesional nestin expression was still apparent 28 days post-injury. Western blot analysis of nestin expression 3 days post-ablation confirmed a significant two-fold increase in nestin expression (p<0.05). Double immunofluorescence labeling demonstrated that the majority of nestin expression occurred in astrocytes. We were unable to detect any colocalization with neuronal makers. However, BrdU-labeled cells, which were readily detected in the subventricular zone prior to injury, were readily detected in the perilesional area 3 days post-ablation, concomitant with nestin in this area. Confocal microscopy detected several BrdU-positive cells expressing nestin. Taken together, the data support a potential role for nestin reexpression in brain repair.

Rescue of Neurons from Ischemic Injury by Peroxisome Proliferator-Activated Receptor-γ Requires a Novel Essential Cofactor LMO4

Activation of peroxisome proliferator-activated receptor-γ (PPARγ) signaling after stroke may reduce brain injury, but this effect will depend on the levels of receptor and cofactors. Here, we showed that the direct effect of PPARγ signaling to protect neurons from ischemic injury requires a novel cofactor LMO4, because this effect was lost in LMO4-null cortical neurons. PPARγ agonist also failed to reduce cerebral infarction after transient focal ischemia in CaMKIIαCre/LMO4loxP mice with LMO4 ablated in neurons of the forebrain. Expressing LMO4 in LMO4-null cortical neurons rescued the PPARγ-protective effect. PPARγ signaling activates the promoter of the antioxidant gene SOD2 and this process requires LMO4. Addition of a superoxide dismutase mimetic MnTBAP [manganese(III)tetrakis(4-benzoic acid)porphyrin] bypassed the deficiency in PPARγ signaling and was able to directly rescue LMO4-null cortical neurons from ischemic injury. Like LMO4, PPARγ and PGC1α (PPARγ coactivator 1α) levels in neurons are elevated by hypoxic stress, and absence of LMO4 impairs their upregulation. Coimmunoprecipitation and mammalian two-hybrid assays revealed that LMO4 interacts in a ligand-dependent manner with PPARγ. LMO4 augments PPARγ-dependent gene activation, in part, by promoting RXRα (retinoid X receptor-α) binding to PPARγ and by increasing PPARγ binding to its target DNA sequence. Together, our results identify LMO4 as an essential hypoxia-inducible cofactor required for PPARγ signaling in neurons. Thus, upregulation of LMO4 expression after stroke is likely to be an important determinant of neuron survival.

C-reactive protein expression in a rodent model of chronic cerebral hypoperfusion

White matter lesions (WML) are a clinically significant, common feature of the aging brain and have been associated with cognitive decline and depression. They are a manifestation of cerebral small vessel disease, which is associated with the progression of vascular dementia. Recent research has been focused on identifying biomarkers which may have a correlation with WML. Previous population based studies have indicated a relation between the serum level of the acute phase protein, C-reactive protein (CRP), and WML. However no previous studies have demonstrated its expression and relation to WML in brain tissue itself. Here we use the rodent model of permanent bilateral common carotid artery ligation (BCCAL) to assess CRP expression during chronic cerebral hypoperfusion (CCH). Our results show that CRP is up-regulated at the mRNA and protein levels in brain tissue from BCCAL animals. The expression of CRP mRNA was upregulated on day 3 following surgery. Because previous studies, as well as the present study, have shown that microglial activity is prominent after the third day of CCH, we sought to determine the role of microglia in CRP expression. Results indicate that cultured microglia express mRNA and protein for CRP and this expression is increased when cells are treated with interleukin-1β (IL-1β), interleukin-6 (IL-6) or a combination of the two.. This finding could indicate a possible role for CRP in the progression of small vessel disease in the brain and provide a therapeutic target.

Regulation of expression of early growth response transcription factors in rat primary cortical neurons by extracellular ATP.

The zinc finger transcription factor early growth response-1 (Egr-1, NGFI-A, zif268, Krox 24, TIS8, ZENK) is upregulated immediately in the brain by cortical spreading depression (CSD) and other preconditioning stimuli and thus might participate in regulation of the overall genomic response to preconditioning. In the present study, the induction of expression of Egr-1 and other early growth response family members was characterized in rat primary cortical neuronal cultures. In neuronal cultures in vitro, depolarization or exposure to extracellular glutamate caused a 4-fold increase in egr-1 mRNA while exposure to extracellular ATP caused a 10-fold increase. The presence of mRNA encoding for multiple types of purinergic receptors was confirmed by RT-PCR. A number of nucleotide agonists proved effective in eliciting an increase in egr-1 mRNA. Over a limited range of concentration, the most effective agonists were ATP > ADP > alpha, beta-methylene ATP > UTP > cAMP > UDP > AMP > adenosine. Pertussis toxin, suramin, reactive blue 2, PPADS, DPCPX and inhibitors of Protein Kinase C, Protein Kinase A and PI3 kinase significantly reduced the upregulation of egr-1 by exposure to extracellular ATP. These findings suggest that neuronal metabotropic purinergic receptor activation contributes to the induction of early growth response transcription factors and may provide a target that can be manipulated to increase ischemic tolerance of the brain in vivo.