Carsten Culmsee

Roles of Nuclear Factor κB in Neuronal Survival and Plasticity

Abstract: The transcription factor nuclear factor κB (NF‐κB) is moving to the forefront of the fields of apoptosis and neuronal plasticity because of recent findings showing that activation of NF‐κB prevents neuronal apoptosis in various cell culture and in vivo models and because NF‐κB is activated in association with synaptic plasticity. Activation of NF‐κB was first shown to mediate antiapoptotic actions of tumor necrosis factor in cultured neurons and was subsequently shown to prevent death of various nonneuronal cells. NF‐κB is activated by several cytokines and neurotrophic factors and in response to various cell stressors. Oxidative stress and elevation of intracellular calcium levels are particularly important inducers of NF‐κB activation. Activation of NF‐κB can interrupt apoptotic biochemical cascades at relatively early steps, before mitochondrial dysfunction and oxyradical production. Gene targets for NF‐κB that may mediate its anti‐apoptotic actions include the antioxidant enzyme manganese superoxide dismutase, members of the inhibitor of apoptosis family of proteins, and the calcium‐binding protein calbindin D28k. NF‐κB is activated by synaptic activity and may play important roles in the process of learning and memory. The available data identify NF‐κB as an important regulator of evolutionarily conserved biochemical and molecular cascades designed to prevent cell death and promote neuronal plasticity. Because NF‐κB may play roles in a range of neurological disorders that involve neuronal degeneration and/or perturbed synaptic function, pharmacological and genetic manipulations of NF‐κB signaling are being developed that may prove valuable in treating disorders ranging from Alzheimer’s disease to schizophrenia.

Glutathione Peroxidase 4 Senses and Translates Oxidative Stress into 12/15-Lipoxygenase Dependent- and AIF-Mediated Cell Death

Oxidative stress in conjunction with glutathione depletion has been linked with various acute and chronic degenerative disorders, yet the molecular mechanisms have remained unclear. In contrast to the belief that oxygen radicals are detrimental to cells and tissues by unspecific oxidation of essential biomolecules, we now demonstrate that oxidative stress is sensed and transduced by glutathione peroxidase 4 (GPx4) into a-yet-unrecognized cell-death pathway. Inducible GPx4 inactivation in mice and cells revealed 12/15-lipoxygenase-derived lipid peroxidation as specific downstream event, triggering apoptosis-inducing factor (AIF)-mediated cell death. Cell death could be entirely prevented either by alpha-tocopherol (alpha-Toc), 12/15-lipoxygenase inhibitors, or siRNA-mediated AIF silencing. Accordingly, 12/15-lipoxygenase-deficient cells were highly resistant to glutathione depletion. Neuron-specific GPx4 depletion caused neurodegeneration in vivo and ex vivo, highlighting the importance of this pathway in neuronal cells. Since oxidative stress is common in the etiology of many human disorders, the identified pathway reveals promising targets for future therapies.

Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable.

AMP-activated protein kinase is highly expressed in neurons in the developing rat brain and promotes neuronal survival following glucose deprivation.

Adenosine monophosphate-activated protein kinase (AMPK) is a member of metabolite-sensing kinase family that plays important roles in responses of muscle cells to metabolic stress. AMPK is a heterotrimer of a catalytic α subunit (α1 or α2), and β (β1 or β2) and γ (γ1 or γ2) subunits. Because the brain has a high metabolic rate and is sensitive to changes in the supply of glucose and oxygen, we investigated the expression of AMPK in rat embryonic and adult brain and its role in modifying neuronal survival under conditions of cellular stress. We report that catalytic (α1 and α2) and noncatalytic (β2 and γ1) subunits of AMPK are present at high levels in embryonic hippocampal neurons in vivo and in cell culture. In the adult rat brain, the catalytic subunits α1 and α2 are present in neurons throughout the brain. The AMPK-activating agent AICAR protected hippocampal neurons against death induced by glucose deprivation, chemical hypoxia, and exposure to glutamate and amyloid β-peptide. Suppression of levels of the AMPK α1 and α2 subunits using antisense technology resulted in enhanced neuronal death following glucose deprivation, and abolished the neuroprotective effect of AICAR. These findings suggest that AMPK can protect neurons against metabolic and excitotoxic insults relevant to the pathogenesis of several different neurodegenerative conditions.

Cellular and Molecular Mechanisms Underlying Perturbed Energy Metabolism and Neuronal Degeneration in Alzheimer's and Parkinson's Diseases

ABSTRACT: Synaptic degeneration and death of nerve cells are defining features of Alzheimers disease (AD) and Parkinsons disease (PD), the two most prevalent age‐related neurodegenerative disorders. In AD, neurons in the hippocampus and basal forebrain (brain regions that subserve learning and memory functions) are selectively vulnerable. In PD dopamine‐producing neurons in the substantia nigra‐striatum (brain regions that control body movements) selectively degenerate. Studies of postmortem brain tissue from AD and PD patients have provided evidence for increased levels of oxidative stress, mitochondrial dysfunction and impaired glucose uptake in vulnerable neuronal populations. Studies of animal and cell culture models of AD and PD suggest that increased levels of oxidative stress (membrane lipid peroxidation, in particular) may disrupt neuronal energy metabolism and ion homeostasis, by impairing the function of membrane ion‐motive ATPases and glucose and glutamate transporters. Such oxidative and metabolic compromise may thereby render neurons vulnerable to excitotoxicity and apoptosis. Studies of the pathogenic mechanisms of AD‐linked mutations in amyloid precursor protein (APP) and presenilins strongly support central roles for perturbed cellular calcium homeostasis and aberrant proteolytic processing of APP as pivotal events that lead to metabolic compromise in neurons. Specific molecular “players” in the neurodegenerative processes in AD and PD are being identified and include Par‐4 and caspases (bad guys) and neurotrophic factors and stress proteins (good guys). Interestingly, while studies continue to elucidate cellular and molecular events occurring in the brain in AD and PD, recent data suggest that both AD and PD can manifest systemic alterations in energy metabolism (e.g., increased insulin resistance and dysregulation of glucose metabolism). Emerging evidence that dietary restriction can forestall the development of AD and PD is consistent with a major “metabolic” component to these disorders, and provides optimism that these devastating brain disorders of aging may be largely preventable.

A synthetic inhibitor of p53 protects neurons against death induced by ischemic and excitotoxic insults, and amyloid β‐peptide

The tumor suppressor protein p53 is essential for neuronal death in several experimental settings and may participate in human neurodegenerative disorders. Based upon recent studies characterizing chemical inhibitors of p53 in preclinical studies in the cancer therapy field, we synthesized the compound pifithrin‐α and evaluated its potential neuroprotective properties in experimental models relevant to the pathogenesis of stroke and neurodegenerative disorders. Pifithrin‐α protected neurons against apoptosis induced by DNA‐damaging agents, amyloid β‐peptide and glutamate. Protection by pifithrin‐α was correlated with decreased p53 DNA‐binding activity, decreased expression of the p53 target gene Bax and suppression of mitochondrial dysfunction and caspase activation. Mice given pifithrin‐α exhibited increased resistance of cortical and striatal neurons to focal ischemic injury and of hippocampal neurons to excitotoxic damage. These preclinical studies demonstrate the efficacy of a p53 inhibitor in models of stroke and neurodegenerative disorders, and suggest that drugs that inhibit p53 may reduce the extent of brain damage in related human neurodegenerative conditions.

Apoptosis-Inducing Factor Triggered by Poly(ADP-Ribose) Polymerase and Bid Mediates Neuronal Cell Death after Oxygen-Glucose Deprivation and Focal Cerebral Ischemia

Delayed neuronal cell death occurring hours after reperfusion is a hallmark of ischemic stroke and a primary target for neuroprotective strategies. In the present study, we investigated whether apoptosis-inducing factor (AIF), a caspase-independent proapoptotic protein, is responsible for neuronal cell death after glutamate toxicity and oxygen-glucose deprivation (OGD) in vitro and after experimental stroke in vivo. AIF translocated to the nucleus in which it colocalized with DNA fragmentation and nuclear apoptotic morphology after exposure to glutamate or OGD in cultured neurons or after transient middle cerebral artery occlusion (MCAo) in mice. Small inhibitory RNA-mediated downregulation of AIF reduced glutamate- and OGD-induced neuronal apoptosis by 37 and 60%, respectively (p < 0.01). Moreover, Harlequin mutant mice, which express AIF at low levels (∼20% of wild-type mice), displayed smaller infarct volumes (-43%; p < 0.03) and showed dramatically reduced cell death in the ischemic penumbra after 45 min of MCAo compared with wild-type littermates. Inhibition of poly(ADP-ribose) polymerase and Bid reduced nuclear AIF translocation. These results provide the first evidence for a causal role of AIF in ischemic neuronal cell death. Therefore, caspase-independent cell death signaling may provide a promising novel target for therapeutic interventions in cerebrovascular diseases.

Neurodegenerative disorders and ischemic brain diseases

Degeneration and death of neurons is the fundamental process responsible for the clinical manifestations of many different neurological disorders of aging, incuding Alzheimers disease, Parkinsons disease and stroke. The death of neurons in such disorders involves apoptotic biochemical cascades involving upstream effectors (Par-4, p53 and pro-apoptotic Bcl-2 family members), mitochondrial alterations and caspase activation. Both genetic and environmental factors, and the aging process itself, contribute to intiation of such neuronal apoptosis. For example, mutations in the amyloid precursor protein and presenilin genes can cause Alzheimers disease, while head injury is a risk factor for both Alzheimers and Parkinsons diseases. At the cellular level, neuronal apoptosis in neurodegenerative disorders may be triggered by oxidative stress, metabolic compromise and disruption of calcium homeostasis. Neuroprotective (anti-apoptotic) signaling pathways involving neurotrophic factors, cytokines and “conditioning responses” can counteract the effects of aging and genetic predisposition in experimental models of neurodegenerative disorders. A better understanding of the molecular underpinnings of neuronal death is leading directly to novel preventative and therapeutic approaches to neurodegenerative disorders.

Transforming Growth Factor-β1 Increases Bad Phosphorylation and Protects Neurons Against Damage

Despite the characterization of neuroprotection by transforming growth factor-β1 (TGF-β1), the signaling pathway mediating its protective effect is unclear. Bad is a proapoptotic member of the Bcl-2 family and is inactivated on phosphorylation via mitogen-activated protein kinase (MAPK). This study attempted to address whether MAPK signaling and Bad phosphorylation were influenced by TGF-β1 and, furthermore, whether these two events were involved in the antiapoptotic effect of TGF-β1. We found a gradual activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and MAPK-activated protein kinase-1 (also called Rsk1) and a concomitant increase in Bad phosphorylation at Ser112 in mouse brains after adenovirus-mediated TGF-β1 transduction under nonischemic and ischemic conditions induced by transient middle cerebral artery occlusion. Consistent with these effects, the ischemia-induced increase in Bad protein level and caspase-3 activation were suppressed in TGF-β1-transduced brain. Consequently, DNA fragmentation, ischemic lesions, and neurological deficiency were significantly reduced. In cultured rat hippocampal cells, TGF-β1 inhibited the increase in Bad expression caused by staurosporine. TGF-β1 concentration- and time-dependently activated Erk1/2 and Rsk1 accompanied by an increase in Bad phosphorylation. These effects were blocked by U0126, a mitogen-activated protein kinase/Erk kinase 1/2 inhibitor, suggesting an association between Bad phosphorylation and MAPK activation. Notably, U0126 and a Rsk1 inhibitor (Ro318220) abolished the neuroprotective activity of TGF-β1 in staurosporine-induced apoptosis, indicating that activation of MAPK is necessary for the antiapoptotic effect of TGF-β1 in cultured hippocampal cells. Together, we demonstrate that TGF-β1 suppresses Bad expression under lesion conditions, increases Bad phosphorylation, and activates the MAPK/Erk pathway, which may contribute to its neuroprotective activity.

Apoptosis-inducing factor is a major contributor to neuronal loss induced by neonatal cerebral hypoxia-ischemia

Nine-day-old harlequin (Hq) mice carrying the hypomorphic apoptosis-inducing factor (AIF)Hq mutation expressed 60% less AIF, 18% less respiratory chain complex I and 30% less catalase than their wild-type (Wt) littermates. Compared with Wt, the infarct volume after hypoxia-ischemia (HI) was reduced by 53 and 43% in male (YXHq) and female (XHqXHq) mice, respectively (P<0.001). The Hq mutation did not inhibit HI-induced mitochondrial release of cytochrome c or activation of calpain and caspase-3. The broad-spectrum caspase inhibitor quinoline-Val-Asp(OMe)-CH2-PH (Q-VD-OPh) decreased the activation of all detectable caspases after HI, both in Wt and Hq mice. Q-VD-OPh reduced the infarct volume equally in Hq and in Wt mice, and the combination of Hq mutation and Q-VD-OPh treatment showed an additive neuroprotective effect. Oxidative stress leading to nitrosylation and lipid peroxidation was more pronounced in ischemic brain areas from Hq than Wt mice. The antioxidant edaravone decreased oxidative stress in damaged brains, more pronounced in the Hq mice, and further reduced brain injury in Hq but not in Wt mice. Thus, two distinct strategies can enhance the neuroprotection conferred by the Hq mutation, antioxidants, presumably compensating for a defect in AIF-dependent redox detoxification, and caspase inhibitors, presumably interrupting a parallel pathway leading to cellular demise.