David C. Henshall

Increased hippocampal neurogenesis in Alzheimer's disease

Neurogenesis, which persists in the adult mammalian brain, may provide a basis for neuronal replacement therapy in neurodegenerative diseases like Alzheimers disease (AD). Neurogenesis is increased in certain acute neurological disorders, such as ischemia and epilepsy, but the effect of more chronic neurodegenerations is uncertain, and some animal models of AD show impaired neurogenesis. To determine how neurogenesis is affected in the brains of patients with AD, we investigated the expression of immature neuronal marker proteins that signal the birth of new neurons in the hippocampus of AD patients. Compared to controls, Alzheimers brains showed increased expression of doublecortin, polysialylated nerve cell adhesion molecule, neurogenic differentiation factor and TUC-4. Expression of doublecortin and TUC-4 was associated with neurons in the neuroproliferative (subgranular) zone of the dentate gyrus, the physiological destination of these neurons (granule cell layer), and the CA1 region of Ammons horn, which is the principal site of hippocampal pathology in AD. These findings suggest that neurogenesis is increased in AD hippocampus, where it may give rise to cells that replace neurons lost in the disease, and that stimulating hippocampal neurogenesis might provide a new treatment strategy.

To die or not to die for neurons in ischemia, traumatic brain injury and epilepsy : a review on the stress activated signaling pathways and apoptotic pathways

After a severe episode of ischemia, traumatic brain injury (TBI) or epilepsy, it is typical to find necrotic cell death within the injury core. In addition, a substantial number of neurons in regions surrounding the injury core have been observed to die via the programmed cell death (PCD) pathways due to secondary effects derived from the various types of insults. Apart from the cell loss in the injury core, cell death in regions surrounding the injury core may also contribute to significant losses in neurological functions. In fact, it is the injured neurons in these regions around the injury core that treatments are targeting to preserve. In this review, we present our cumulated understanding of stress-activated signaling pathways and apoptotic pathways in the research areas of ischemic injury, TBI and epilepsy and that gathered from concerted research efforts in oncology and other diseases. However, it is obvious that our understanding of these pathways in the context of acute brain injury is at its infancy stage and merits further investigation. Hopefully, this added research effort will provide a more detailed knowledge from which better therapeutic strategies can be developed to treat these acute brain injuries.

Silencing microRNA-134 produces neuroprotective and prolonged seizure-suppressive effects

Temporal lobe epilepsy is a common, chronic neurological disorder characterized by recurrent spontaneous seizures. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate post-transcriptional expression of protein-coding mRNAs, which may have key roles in the pathogenesis of neurological disorders. In experimental models of prolonged, injurious seizures (status epilepticus) and in human epilepsy, we found upregulation of miR-134, a brain-specific, activity-regulated miRNA that has been implicated in the control of dendritic spine morphology. Silencing of miR-134 expression in vivo using antagomirs reduced hippocampal CA3 pyramidal neuron dendrite spine density by 21% and rendered mice refractory to seizures and hippocampal injury caused by status epilepticus. Depletion of miR-134 after status epilepticus in mice reduced the later occurrence of spontaneous seizures by over 90% and mitigated the attendant pathological features of temporal lobe epilepsy. Thus, silencing miR-134 exerts prolonged seizure-suppressant and neuroprotective actions; determining whether these are anticonvulsant effects or are truly antiepileptogenic effects requires additional experimentation.

Neuroprotective Actions of FK506 in Experimental Stroke: In Vivo Evidence against an Antiexcitotoxic Mechanism

The cellular mechanisms underlying the neuroprotective action of the immunosuppressant FK506 in experimental stroke remain uncertain, although in vitro studies have implicated an antiexcitotoxic action involving nitric oxide and calcineurin. The present in vivo study demonstrates that intraperitoneal pretreatment with 1 and 10 mg/kg FK506, doses that reduced the volume of ischemic cortical damage by 56–58%, did not decrease excitotoxic damage induced by quinolinate, NMDA, and AMPA. Similarly, intravenous FK506 did not reduce the volume of striatal quinolinate lesions at a dose (1 mg/kg) that decreased ischemic cortical damage by 63%. The temporal window for FK506 neuroprotection was defined in studies demonstrating efficacy using intravenous administration at 120 min, but not 180 min, after middle cerebral artery occlusion. The noncompetitive NMDA receptor antagonist MK801 reduced both ischemic and excitotoxic damage. Histopathological data concerning striatal quinolinate lesions were replicated in neurochemical experiments. MK801, but not FK506, attenuated the loss of glutamate decarboxylase and choline acetyltransferase activity induced by intrastriatal injection of quinolinate. The contrasting efficacy of FK506 in ischemic and excitotoxic lesion models cannot be explained by drug pharmacokinetics, because brain FK506 content rose rapidly using both treatment protocols and was sustained at a neuroprotective level for 3 d. Although these data indicate that an antiexcitotoxic mechanism is unlikely to mediate the neuroprotective action of FK506 in focal cerebral ischemia, the finding that intravenous cyclosporin A (20 mg/kg) reduced ischemic cortical damage is consistent with the proposed role of calcineurin.

Endotoxin Preconditioning Prevents Cellular Inflammatory Response During Ischemic Neuroprotection in Mice

Background and Purpose— Tolerance to ischemic brain injury is induced by several preconditioning stimuli, including lipopolysaccharide (LPS). A small dose of LPS given systemically confers ischemic protection in the brain, a process that appears to involve activation of an inflammatory response before ischemia. We postulated that LPS preconditioning modulates the cellular inflammatory response after cerebral ischemia, resulting in neuroprotection. Methods— Mice were treated with LPS (0.2 mg/kg) 48 hours before ischemia induced by transient middle cerebral artery occlusion (MCAO). The infarct was measured by 2,3,5-triphenyltetrazolium chloride staining. Microglia/macrophage responses after MCAO were assessed by immunofluorescence and flow cytometry. The effect of MCAO on white blood cells in the brain and peripheral circulation was measured by flow cytometry 48 hours after MCAO. Results— LPS preconditioning induced significant neuroprotection against MCAO. Administration of low-dose LPS before MCAO prevented the cellular inflammatory response in the brain and blood. Specifically, LPS preconditioning suppressed neutrophil infiltration into the brain and microglia/macrophage activation in the ischemic hemisphere, which was paralleled by suppressed monocyte activation in the peripheral blood. Conclusions— LPS preconditioning induces neuroprotection against ischemic brain injury in a mouse model of stroke. LPS preconditioning suppresses the cellular inflammatory response to ischemia in the brain and circulation. Diminished activation of cellular inflammatory responses that ordinarily exacerbate ischemic injury may contribute to neuroprotection induced by LPS preconditioning.

CREB-mediated Bcl-2 protein expression after ischemic preconditioning

Bcl-2 plays a pivotal role in the control of cell death and is upregulated by ischemic tolerance. Because Bcl-2 expression is regulated by the transcription factor cyclic AMP response element-binding protein (CREB), we investigated the role of CREB activation in two models of ischemic preconditioning: focal ischemic tolerance after middle cerebral artery occlusion (MCAO) and in vitro ischemic tolerance modeled by oxygen–glucose deprivation (OGD). After preconditioning ischemia (30 minutes MCAO or 30 minutes OGD), phosphorylation of CREB was increased, and there was an increased interaction between the bcl-2 cyclic AMP-responsive element (CRE) promoter and nuclear proteins after preconditioning ischemia in vivo and in vitro. Chromatin immunoprecipitation revealed an increased interaction between CREB-binding protein and the bcl-2 CRE rather than CREB, after preconditioning ischemia. Ischemic tolerance was blocked by a CRE decoy oligonucleotide, which also blocked Bcl-2 expression. The protein kinase A inhibitor H89, the calcium/calmodulin kinase inhibitor KN62, and the MEK inhibitor U0126 blocked ischemic tolerance, but not the phosphatidylinositol 3-kinase inhibitor LY294002. H89, KN62, and U0126 reduced CREB activation and Bcl-2 expression. Taken together, these data suggest that after ischemic preconditioning CREB activation regulates the expression of the prosurvival protein Bcl-2.

Epilepsy and Apoptosis Pathways

Epilepsy is a common, chronic neurologic disorder characterized by recurrent unprovoked seizures. Experimental modeling and clinical neuroimaging of patients has shown that certain seizures are capable of causing neuronal death. Such brain injury may contribute to epileptogenesis, impairments in cognitive function or the epilepsy phenotype. Research into cell death after seizures has identified the induction of the molecular machinery of apoptosis. Here, the authors review the clinical and experimental evidence for apoptotic cell death pathway function in the wake of seizure activity. We summarize work showing intrinsic (mitochondrial) and extrinsic (death receptor) apoptotic pathway function after seizures, activation of the caspase and Bcl-2 families of cell death modulators and the acute and chronic neuropathologic impact of intervening in these molecular cascades. Finally, we describe evolving data on nonlethal roles for these proteins in neuronal restructuring and cell excitability that have implications for shaping the epilepsy phenotype. This review highlights the work to date on apoptosis pathway signaling during seizure-induced neuronal death and epileptogenesis, and speculates on how emerging roles in brain remodeling and excitability have enriched the number of therapeutic strategies for protection against seizure-damage and epileptogenesis.

Alterations in bcl-2 and caspase gene family protein expression in human temporal lobe epilepsy

Objective: To address the role of cell death regulatory genes of the bcl-2 and caspase families in the neuropathology of human epilepsy using tissue extracted from patients undergoing temporal lobectomy for intractable seizures. Methods: Using Western blotting and immunohistochemistry, the authors investigated the expression of bcl-2, bcl-xL, bax, caspase-1, and caspase-3 in temporal cortex samples from patients who had undergone temporal lobectomy surgery for intractable epilepsy (n = 19). Nonepileptic postmortem tissue from a brain bank served as control (n = 6). Results: Western blot analysis demonstrated significant increases in levels of bcl-2 and bcl-xL protein in seizure brain compared to control. Cleavage of caspase-1 was evidenced by a reduction in levels of the 45 kDa proenzyme form and an increase in levels of the p10 fragment. Levels of the 32 kDa proenzyme form of caspase-3 were elevated in seizure patients, as were levels of the 12 kDa cleaved fragment. Bcl-2, bax, and caspase-3 immunoreactivity was increased predominantly in cells with the morphologic appearance of neurons, whereas bcl-xL immunoreactivity was increased in cells with the appearance of glia. DNA fragmentation was detected in some but not all sections from epileptic brain samples. Conclusions: Cell death regulatory genes of the bcl-2 and caspase families may play a role in ongoing neuropathologic processes in human epilepsy, and offer novel targets as an adjunct to anticonvulsant therapy.

Involvement of caspase-3-like protease in the mechanism of cell death following focally evoked limbic seizures

The cysteine protease caspase‐3 may be involved in the mechanism of cell death following seizures. Using a rat model of focally evoked limbic epilepsy with continuous electroencephalography monitoring, we investigated seizure‐induced changes in caspase‐3 protein expression and processing, enzyme activity, and the in vivo effect of caspase‐3 inhibition. Seizures were induced by intraamygdaloid injection of kainic acid (0.1 μg) and were terminated after 45 min by diazepam (30 mg/kg) administration. Animals were killed 0‐72 h following diazepam administration. Levels of the 32‐kDa proenzyme form of caspase‐3 were unaffected by seizures. Levels of the 17‐kDa cleaved (active) fragment of caspase‐3 were almost undetectable in control brain, but were increased significantly at 4 and 24 h within ipsilateral hippocampus and cortex in seizure animals. Caspase‐3‐like protease activity was increased within the ipsilateral hippocampus at 8 and 24 h following seizures. Caspase‐3 immunoreactivity was increased within the vulnerable ipsilateral CA3/CA4 subfield at 24 and 72 h following seizures and was associated predominantly, but not exclusively, with neurons exhibiting DNA fragmentation. The putatively selective caspase‐3 inhibitor N‐benzyloxycarbonyl‐Asp(OMe)‐Glu(OMe)‐Val‐Asp(OMe)‐fluoromethyl ketone significantly improved neuronal survival bilaterally within the hippocampal CA3/CA4 subfields following seizures. Collectively, these data suggest that caspase‐3 may play a significant role in the mechanism by which neurons die following seizures.

Endotoxin preconditioning protects against the cytotoxic effects of TNFα after stroke: A novel role for TNFα in LPS-ischemic tolerance

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury. Tumor necrosis factor-α (TNFα) is protective in LPS-induced preconditioning yet exacerbates neuronal injury in ischemia. Here, we define dual roles of TNFα in LPS-induced ischemic tolerance in a murine model of stroke and in primary neuronal cultures in vitro, and show that the cytotoxic effects of TNFα are attenuated by LPS preconditioning. We show that LPS preconditioning significantly increases circulating levels of TNFα before middle cerebral artery occlusion in mice and show that TNFα is required to establish subsequent neuroprotection against ischemia, as mice lacking TNFα are not protected from ischemic injury by LPS preconditioning. After stroke, LPS preconditioned mice have a significant reduction in the levels of TNFα (~ threefold) and the proximal TNFα signaling molecules, neuronal TNF-receptor 1 (TNFR1), and TNFR-associated death domain (TRADD). Soluble TNFR1 (s-TNFR1) levels were significantly increased after stroke in LPS-preconditioned mice (~ 2.5-fold), which may neutralize the effect of TNFα and reduce TNFα-mediated injury in ischemia. Importantly, LPS-preconditioned mice show marked resistance to brain injury caused by intracerebral administration of exogenous TNFα after stroke. We establish an in vitro model of LPS preconditioning in primary cortical neuronal cultures and show that LPS preconditioning causes significant protection against injurious TNFα in the setting of ischemia. Our studies suggest that TNFα is a twin-edged sword in the setting of stroke: TNFα upregulation is needed to establish LPS-induced tolerance before ischemia, whereas suppression of TNFα signaling during ischemia confers neuroprotection after LPS preconditioning.